Thraustochytrids, fatty acid compositions, and methods of making and uses thereof

ABSTRACT

The present invention is directed to isolated thraustochytrid microorganisms as well as strains and mutants thereof. The invention is further directed to biomasses, microbial oils, compositions, cultures, methods of producing microbial oils, and methods of using the isolated thraustochytrids, biomasses, and microbial oils.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (“sequencelisting.txt”, 10,531 bytes, created on Feb. 26, 2009) filed with theapplication is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to isolated thraustochytridmicroorganisms as well as strains and mutants thereof. The invention isfurther directed to thraustochytrid biomasses, microbial oils,compositions, cultures, methods of producing the microbial oils, andmethods of using the isolated thraustochytrids, biomasses, and microbialoils.

2. Background Art

Fatty acids are classified based on the length and saturationcharacteristics of the carbon chain. Short chain fatty acids generallyhave 12 carbons or less, medium chain fatty acids generally have 14 to18 carbons, and long chain fatty acids generally have 20 or morecarbons. Fatty acids are termed saturated fatty acids when no doublebonds are present between the carbon atoms and are termed unsaturatedfatty acids when double bonds are present. Unsaturated long chain fattyacids are monounsaturated when only one double bond is present and arepolyunsaturated when more than one double bond is present.

Polyunsaturated fatty acids (PUFAs) are classified based on the positionof the first double bond from the methyl end of the fatty acid: omega-3(n-3) fatty acids contain a first double bond at the third carbon, whileomega-6 (n-6) fatty acids contain a first double bond at the sixthcarbon. For example, docosahexaenoic acid (“DHA”) is an omega-3 longchain polyunsaturated fatty acid (LC-PUFA) with a chain length of 22carbons and 6 double bonds, often designated as “22:6 n-3.” Otheromega-3 LC-PUFAs include eicosapentaenoic acid (“EPA”), designated as“20:5 n-3,” and omega-3 docosapentaenoic acid (“DPA n-3”), designated as“22:5 n-3.” DHA and EPA have been termed “essential” fatty acids.Omega-6 LC-PUFAs include arachidonic acid (“ARA”), designated as “20:4n-6,” and omega-6 docosapentaenoic acid (“DPA n-6”), designated as “22:5n-6.”

Omega-3 fatty acids are biologically important molecules that affectcellular physiology due to their presence in cell membranes, regulateproduction and gene expression of biologically active compounds, andserve as biosynthetic substrates. Roche, H. M., Proc. Nutr. Soc. 58:397-401 (1999). DHA, for example, accounts for approximately 15%-20% oflipids in the human cerebral cortex, 30%-60% of lipids in the retina, isconcentrated in the testes and sperm, and is an important component ofbreast milk. Jean-Pascal Bergé & Gilles Barnathan, Fatty Acids fromLipids of Marine Organisms: Molecular Biodiversity, Roles as Biomarkers,Biologically Active Compounds, and Economical Aspects, in MarineBiotechnology 149 (T. Scheper, ed., 2005). DHA accounts for up to 97% ofthe omega-3 fatty acids in the brain and up to 93% of the omega-3 fattyacids in the retina. Moreover, DHA is essential for both fetal andinfant development as well as maintenance of cognitive functions inadults. Id. Omega-3 fatty acids, including DHA and EPA, also possessanti-inflammatory properties. See, e.g., Id. and Simopoulos, A. P., J.Am. Coll. Nutr. 21: 495-595 (2002). Because omega-3 fatty acids are notsynthesized de novo in the human body, these fatty acids must be derivedfrom nutritional sources.

Flaxseed oil and fish oils are considered good dietary sources ofomega-3 fatty acids. Flaxseed oil contains no EPA, DHA, DPA, or ARA butrather contains linolenic acid (C18:3 n-3), a building block enablingthe body to manufacture EPA. There is evidence, however, that the rateof metabolic conversion can be slow and variable, particularly amongthose with impaired health. Fish oils vary considerably in the type andlevel of fatty acid composition depending on the particular species andtheir diets. For example, fish raised by aquaculture tend to have alower level of omega-3 fatty acids than those in the wild. Furthermore,fish oils carry the risk of containing environmental contaminants andcan be associated with stability problems and a fishy odor or taste.

Thraustochytrids are microorganisms of the order Thraustochytriales.Thraustochytrids include members of the genus Schizochytrium and havebeen recognized as an alternative source of omega-3 fatty acids,including DHA. See U.S. Pat. No. 5,130,242. Oils produced from thesemarine heterotrophic microorganisms often have simpler polyunsaturatedfatty acid profiles than corresponding fish or microalgal oils. Lewis,T. E., Mar. Biotechnol. 1: 580-587 (1999). Strains of thraustrochytridspecies have been reported to produce omega-3 fatty acids as a highpercentage of the total fatty acids produced by the organisms. U.S. Pat.No. 5,130,242; Huang, J. et al., J. Am. Oil. Chem. Soc. 78: 605-610(2001); Huang, J. et al., Mar. Biotechnol. 5: 450-457 (2003). However,isolated thraustochytrids vary in the identity and amounts of LC-PUFAsproduced, such that some previously described strains can haveundesirable levels of omega-6 fatty acids and/or can demonstrate lowproductivity in culture. As such, a continuing need exists for theisolation of thraustochytrids demonstrating high productivity anddesirable LC-PUFA profiles.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to an isolated thraustochytridmicroorganism of the thraustochytrid species deposited under ATCCAccession No. PTA-9695 or a strain derived therefrom, wherein the totalfatty acids produced by said microorganism or strain derived therefromcomprise about 10% or less by weight eicosapentaenoic acid.

The present invention is also directed to an isolated thraustochytridmicroorganism having the characteristics of the thraustochytrid speciesdeposited under ATCC Accession No. PTA-9695, wherein the total fattyacids produced by said microorganism or strain derived therefromcomprise about 10% or less by weight eicosapentaenoic acid.

The present invention is also directed to an isolated thraustochytridmicroorganism, or a strain derived therefrom, comprising a triglyceridefraction, wherein the docosahexaenoic acid content of the triglyceridefraction is at least about 40% by weight, wherein the docosapentaenoicacid n-6 content of the triglyceride fraction is from at least about0.5% by weight to about 6% by weight, and wherein the total fatty acidsproduced by said microorganism or strain derived therefrom compriseabout 10% or less by weight eicosapentaenoic acid.

The present invention is also directed to an isolated thraustochytridmicroorganism of the same species as the thraustochytrid deposited underATCC Accession No. PTA-9695, or a strain derived therefrom, wherein thetotal fatty acids produced by said microorganism or strain derivedtherefrom comprise about 10% or less by weight eicosapentaenoic acid.

In some embodiments, the strain derived from the isolatedthraustochytrid microorganism of the invention is a mutant strain.

The present invention is also directed to an isolated microorganismdeposited under ATCC Accession No. PTA-9695, PTA-9696, PTA-9697, orPTA-9698.

The present invention is also directed to a thraustochytrid biomasscomprising any one of the thraustochytrid microorganisms of theinvention or mixtures thereof.

The present invention is also directed to an isolated thraustochytridbiomass, wherein at least about 50% by weight of the dry cell weight ofthe biomass are fatty acids, and wherein at least about 50% by weight ofthe fatty acids are omega-3 fatty acids. In some embodiments, at leastabout 50% by weight of the fatty acids is docosahexaenoic acid. Thepresent invention is also directed to an isolated thraustochytridbiomass, wherein at least about 25% by weight of the dry cell weight ofthe biomass is docosahexaenoic acid.

In some embodiments, the present invention is also directed to anisolated thraustochytrid biomass wherein about 10% or less by weight ofthe fatty acids is eicosapentaenoic acid, and wherein the weight ratioof docosahexaenoic acid to eicosapentaenoic acid is at least about 5:1.

In some embodiments, the present invention is also directed to anisolated thraustochytrid biomass wherein about 1.5% or less by weight ofthe fatty acids is arachidonic acid, and wherein the weight ratio ofdocosahexaenoic acid to arachidonic acid is at least about 20:1.

In some embodiments, the present invention is also directed to anisolated thraustochytrid biomass comprising docosahexaenoic acid anddocosapentaenoic acid n-6 in a weight ratio of at least about 10:1.

The present invention is also directed to an isolated thraustochytridculture comprising any one of the thraustochytrid microorganisms of theinvention or mixtures thereof. In some embodiments, the culturecomprises at least about 5% dissolved oxygen.

The present invention is also directed to a food product, cosmetic, orpharmaceutical composition for animals or humans comprising any one ofthe thraustochytrid microorganisms or biomasses of the invention ormixtures thereof.

The present invention is also directed to a microbial oil comprising atriglyceride fraction of at least about 70% by weight, wherein thedocosahexaenoic acid content of the triglyceride fraction is at leastabout 50% by weight, and wherein the docosapentaenoic acid n-6 contentof the triglyceride fraction is from about 0.5% by weight to about 6% byweight. In some embodiments, the microbial oil further comprises anarachidonic acid content of the triglyceride fraction of about 1.5% orless by weight.

The present invention is also directed to a microbial oil comprising atriglyceride fraction of at least about 70% by weight, wherein thedocosahexaenoic acid content of the triglyceride fraction is at leastabout 40% by weight, wherein the docosapentaenoic acid n-6 content ofthe triglyceride fraction is from at least about 0.5% by weight to about6% by weight, and wherein the ratio of docosahexaenoic acid todocosapentaenoic acid n-6 is greater than about 6:1.

The present invention is also directed to a microbial oil comprising atriglyceride fraction of at least about 70% by weight, wherein thedocosahexaenoic acid content of the triglyceride fraction is at leastabout 60% by weight.

In some embodiments, at least about 20% of the triglycerides in thetriglyceride fraction of the microbial oil contain docosahexaenoic acidat two positions in the triglyceride selected from any two of the sn-1,sn-2, and sn-3 positions. In some embodiments, at least about 5% of thetriglycerides in the triglyceride fraction of the microbial oil containdocosahexaenoic acid at all three of the sn-1, sn-2, and sn-3 positionsin the triglyceride.

In some embodiments, the microbial oil further comprises about 5% orless by weight of heptadecanoic acid.

The present invention is also directed to a food product, cosmetic, orpharmaceutical composition for animals or humans comprising any of themicrobial oils of the invention. In some embodiments, the food productis an infant formula. In some embodiments, the infant formula issuitable for premature infants. In some embodiments, the food product isa milk, a beverage, a therapeutic drink, a nutritional drink, or acombination thereof. In some embodiments, the food product is anadditive for animal or human food. In some embodiments, the food productis a nutritional supplement. In some embodiments, the food product is ananimal feed. In some embodiments, the animal feed is an aquaculturefeed. In some embodiments, the animal feed is a domestic animal feed, azoological animal feed, a work animal feed, a livestock feed, or acombination thereof.

The present invention is also directed to a method for producing amicrobial oil comprising omega-3 fatty acids, the method comprising: (a)growing any one of the isolated thraustochytrid microorganisms of theinvention or mixtures thereof in a culture to produce a biomass, and (b)extracting an oil comprising omega-3 fatty acids from the biomass. Insome embodiments, the culture comprises at least about 5% dissolvedoxygen. In some embodiments, the culture pH is maintained at about 6.5to about 8.5. In some embodiments, the culture temperature is maintainedat about 17° C. to about 30° C. In some embodiments, the culturecomprises a glucose concentration of about 5 g/L to about 50 g/L.

The present invention is also directed to a method for producing amicrobial oil comprising omega-3 fatty acids, the method comprisingextracting an oil comprising omega-3 fatty acids from any one of thebiomasses of the invention. In some embodiments, the microbial oil isextracted using a hexane extraction process. In some embodiments, themicrobial oil is extracted using a solventless extraction process.

In some embodiments, the dry cell weight of the biomass isolated fromeach liter of any one of the cultures of the invention is at least about50 g after growing for 7 days at about 17° C. to about 30° C. in aculture medium of about pH 6.5 to about pH 8.0 comprising sources ofcarbon, nitrogen, and nutrients, and about 950 ppm to about 8500 ppmchloride ions.

In some embodiments, any one of the isolated cultures of the inventionhas an omega-3 fatty acid productivity of at least about 2 g/L/day aftergrowing for about 7 days at about 17° C. to about 30° C. in a culturemedium of about pH 6.5 to about pH 8.0 comprising sources of carbon,nitrogen, and nutrients, and about 950 ppm to about 8500 ppm chlorideions.

The present invention is also directed to a microbial oil produced by amethod of the invention.

The present invention is also directed to use of any of the isolatedmicroorganisms, biomasses, or microbial oils of the invention, ormixtures thereof, for the manufacture of a medicament for the treatmentof inflammation or a condition related thereto.

The present invention is also directed to use of any of the isolatedmicroorganisms, biomasses, or microbial oils of the invention, ormixtures thereof, for the treatment of inflammation or a conditionrelated thereto.

The present invention is also directed to any of the isolatedmicroorganisms, biomasses, or microbial oils of the invention, ormixtures thereof, for use in the treatment of inflammation or acondition related thereto.

The present invention is also directed to a method for treatinginflammation or a condition related thereto in a subject in needthereof, comprising administering to the subject any of the isolatedmicroorganisms, biomasses, or microbial oils of the invention, ormixtures thereof, and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to isolated thraustochytridmicroorganisms, as well as strains and mutants thereof, as well asbiomasses, microbial oils, compositions, and cultures thereof. Thepresent invention is also directed to methods of producing microbialoils from the thraustochytrids of the invention, and methods of usingthe thraustochytrids, biomasses, and microbial oils. Thethraustochytrids described herein are highly productive as compared toprior isolates and produce unique fatty acid profiles, characterized inpart by high levels of omega-3 fatty acids, in particular high levels ofDHA.

Thraustochytrid Microorganisms

The invention is directed to isolated thraustochytrids, includingmutants, recombinants, and variants thereof.

In some embodiments, the invention is directed to a thraustochytrid ofthe species deposited under ATCC Accession No. PTA-9695. The isolatedthraustochytrid is also known herein as Schizochytrium sp. ATCCPTA-9695. The thraustochytrid associated with ATCC Accession No.PTA-9695 was deposited under the Budapest Treaty on Jan. 7, 2009 at theAmerican Type Culture Collection, Patent Depository, 10801 UniversityBoulevard, Manassas, Va. 20110-2209.

In some embodiments, the invention is directed to an isolatedthraustochytrid strain deposited under ATCC Accession No. PTA-9695. Insome embodiments, the invention is directed to an isolatedthraustochytrid microorganism of the same species as the thraustochytriddeposited under ATCC Accession No. PTA-9695.

In some embodiments, the invention is directed to an isolatedthraustochytrid having the characteristics of the species depositedunder ATCC Accession No. PTA-9695 or a strain derived therefrom. Thecharacteristics of the thraustochytrid species deposited under ATCCAccession No. PTA-9695 include its growth and phenotypic properties(examples of phenotypic properties include morphological andreproductive properties), its physical and chemical properties (such asdry weights and lipid profiles), and its gene sequences. In someembodiments, the isolated thraustochytrids of the invention havesubstantially identical phenotypic properties of the thraustochytriddeposited under ATCC Accession No. PTA-9695. In some embodiments, theisolated thraustochytrids of the invention have substantially identicalgrowth properties of the thraustochytrid deposited under ATCC AccessionNo. PTA-9695.

In some embodiments, the invention is directed to a mutant, variant, orrecombinant of an isolated thraustochytrid of the invention, wherein thetotal fatty acids produced by the mutant, variant, or recombinantcomprise about 10% or less by weight eicosapentaenoic acid. Mutantstrains can be produced by well-known procedures. Common proceduresinclude irradiation; treatment at high temperatures; and treatment witha mutagen. Variant strains can be other naturally occurring isolatesand/or subisolates of the species described herein. Recombinant strainscan be produced by any well-known methods in molecular biology for theexpression of exogenous genes or the alteration of endogenous genefunction or expression. In some embodiments, the mutant, variant, orrecombinant strain produces a higher amount of omega-3 fatty acids,including DHA and/or EPA, than the wild-type strain. In someembodiments, the mutant, variant, or recombinant strain produces a loweramount of one or more fatty acids, such as lower amounts of EPA, ARA,DPA n-6, or combinations thereof. In some embodiments, the mutant,variant, or recombinant strain produces a larger dry cell weight perliter of culture than the wild-type strain. Such mutant, variant, orrecombinant strains are examples of strains derived from an isolatedthraustochytrid of the invention.

In some embodiments, the invention is directed to a mutant strain of thethraustochytrid deposited under ATCC Accession No. PTA-9695. In furtherembodiments, the mutant strain is a strain deposited under ATCCAccession Nos. PTA-9696, PTA-9697, or PTA-9698. The thraustochytridstrains associated with ATCC Accession Nos. PTA-9696, PTA-9697, andPTA-9698 were deposited under the Budapest Treaty on Jan. 7, 2009 at theAmerican Type Culture Collection, Patent Depository, UniversityBoulevard, Manassas, Va. 20110-2209. These deposited mutant strains arederivatives of the thraustochytrid deposited under ATCC Accession No.PTA-9695.

In some embodiments, an isolated thraustochytrid of the invention,including mutants, variants, or recombinants thereof, comprises a fattyacid profile in one or more fractions isolated from the thraustochytrid.The one or more fractions isolated from the thraustochytrid includes thetotal fatty acid fraction, the sterol esters fraction, the triglyceridefraction, the free fatty acid fraction, the sterol fraction, thediglyceride fraction, the polar fraction (including the phospholipidfraction), and combinations thereof.

Thraustochytrid Cultures and Isolated Thraustochytrid Biomasses

The invention is further directed to a culture comprising one or moreisolated thraustochytrids of the invention. Various fermentationparameters for inoculating, growing, and recovering microflora are knownin the art, such as described in U.S. Pat. No. 5,130,242. Anyconventional medium for growth of thraustochytrids can be used. Liquidor solid mediums can contain natural or artificial sea water. Carbonsources include, but are not limited to, glucose, fructose, xylose,saccharose, maltose, soluble starch, molasses, fucose, glucosamine,dextran, fats, oils, glycerol, sodium acetate, and mannitol. Nitrogensources include, but are not limited to, peptone, yeast extract,polypeptone, malt extract, meat extract, casamino acid, corn steepliquor, organic nitrogen sources, sodium glutamate, urea, inorganicnitrogen sources, ammonium acetate, ammonium sulfate, ammonium chloride,ammonium nitrate, sodium sulfate. A typical media is shown in Table 1:

TABLE 1 Vessel Media Ingredient concentration ranges NaCl g/L 12.5 0-25,5-20, or 10-15 KCl g/L 1.0 0-5, 0.25-3, or 0.5-2 MgSO₄•7H₂O g/L 5.00-10, 2-8, or 3-6 (NH₄)₂SO₄ g/L 0.6 0-10, 0.25-5, or 0.5-3 CaCl₂ g/L0.29 0.1-5, 0.15-3, or 0.2-1 T 154 (yeast extract) g/L 6.0 0-20, 1-15,or 5-10 KH₂PO₄ g/L 1.2 0.1-10, 0.5-5, or 1-3 Post autoclave (Metals)Citric acid mg/L 3.5 0.1-100, 1-50, or 2-25 FeSO₄•7H₂O mg/L 10.300.1-100, 1-50, or 5-25 MnCl₂•4H₂O mg/L 3.10 0.1-100, 1-50, or 2-25ZnSO₄•7H₂O mg/L 3.10 0.1-100, 1-50, or 2-25 CoCl₂•6H₂O mg/L 0.040.001-1, 0.005-0.5, or 0.01-0.1 Na₂MoO₄•2H₂O mg/L 0.04 0.001-1,0.005-0.5, or 0.01-0.1 CuSO₄•5H₂O mg/L 2.07 0.1-100, 0.5-50, or 1-25NiSO₄•6H₂O mg/L 2.07 0.1-100, 0.5-50, or 1-25 Post autoclave (Vitamins)Thiamine** mg/L 9.75 0.1-100, 1-50, or 5-25 Vitamin B12** mg/L 0.160.1-100, 0.1-10, or 0.1-1 Ca½-pantothenate** mg/L 3.33 0.1-100, 0.1-50,or 1-10 Post autoclave (Carbon) Glucose g/L 30.0 5-150, 10-100, or 20-50Nitrogen Feed: NH₄OH mL/L 21.6 0-150, 10-100, or 15-50Typical cultivation conditions would include the following:

pH about 6.5-about 8.5, about 6.5-about 8.0, or about 7.0-about 7.5temperature: about 17-about 30 degrees Celsius, about 20-about 25degrees Celsius, or about 22 to about 23 degrees Celsius dissolvedoxygen: about 5-about 100% saturation, about 10-about 80% saturation, orabout 20-about 50% saturation glucose about 5-about 50 g/L, about10-about 40 g/L, or controlled @: about 20-about 35 g/L.

In some embodiments, the culture medium comprises at least about 5%, atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90% dissolved oxygen, as a percentageof saturation level. In some embodiments, the culture medium comprisesfrom about 5% to about 20%, about 5% to about 50%, about 5% to about100%, about 10% to about 20%, about 10% to about 50%, about 10% to about100%, about 20% to about 50%, or about 20% to about 100% dissolvedoxygen, as a percentage of saturation level.

The invention is further directed to an isolated biomass of athraustochytrid of the invention. An isolated thraustochytrid biomass ofthe invention is a harvested cellular biomass obtained by anyconventional method for the isolation of a thraustochytrid biomass, suchas described in U.S. Pat. No. 5,130,242 and U.S. Appl. Publ. No.2002/0001833.

In some embodiments, the dry cell weight of the biomass isolated fromeach liter of culture is at least about 50 g, at least about 60 g, atleast about 70 g, at least about 80 g, at least about 100 g, at leastabout 120 g, at least about 140 g, at least about 160 g, at least about180 g, or at least about 200 g after growing for about 7 days at about17° C. to about 30° C. in a culture medium of about pH 6.5 to about 8.5comprising sources of carbon, nitrogen, and nutrients, and about 950 ppmto about 8500 ppm chloride ions. In some embodiments, the dry cellweight of the biomass isolated from each liter of culture is at leastabout 50 g, at least about 60 g, at least about 70 g, at least about 80g, at least about 100 g, at least about 120 g, at least about 140 g, atleast about 160 g, at least about 180 g, or at least about 200 g aftergrowing for about 7 days at about 17° C., at about 18° C., at about 19°C., at about 20° C., at about 21° C., at about 22° C., at about 23° C.,at about 24° C., at about 25° C., at about 26° C., at about 27° C., atabout 28° C., at about 29° C., or at about 30° C. in a culture medium ofabout pH 6.5, about pH 7, about pH 7.5, about pH 8.0, or about pH 8.5comprising sources of carbon, nitrogen, and nutrients, and about 950 ppmto about 8500 ppm chloride ions. In some embodiments, the dry cellweight of the biomass isolated from each liter of culture is from about50 g to about 200 g after growing for about 7 days at about 17° C. toabout 30° C. in a culture medium of about pH 6.5 to about pH 8.5comprising sources of carbon, nitrogen, and nutrients, and about 950 ppmto about 8500 ppm chloride ions. In some embodiments, the dry cellweight of the biomass isolated from each liter of culture is from about50 g to about 200 g after growing for about 7 days at about 17° C., atabout 18° C., at about 19° C., at about 20° C., at about 21° C., atabout 22° C., at about 23° C., at about 24° C., at about 25° C., atabout 26° C., at about 27° C., at about 28° C., at about 29° C., or atabout 30° C. in a culture medium of about pH 6.5, about pH 7, about pH7.5, about pH 8.0, or about pH 8.5 comprising sources of carbon,nitrogen, and nutrients, and about 950 ppm to about 8500 ppm chlorideions.

In some embodiments, the isolated thraustochytrid culture has an omega-3fatty acid productivity of at least about 2 g/L/day, at least about 4g/L/day, or at least about 8 g/L/day after growing for about 7 days atabout 17° C. to about 30° C. in a culture medium of about pH 6.5 toabout pH 8.5 comprising sources of carbon, nitrogen, and nutrients, andabout 950 ppm to about 8500 ppm chloride ions. In some embodiments, theisolated thraustochytrid culture has an omega-3 fatty acid productivityof between about 1 g/L/day to about 20 g/L/day, about 2 g/L/day to about15 g/L/day, about 2 g/L/day to about 10 g/L/day, about 3 g/L/day toabout 10 g/L/day, or about 4 g/L/day to about 9 g/L/day, after growingfor about 7 days at about 17° C. to about 30° C. in a culture medium ofabout pH 6.5 to about pH 8.5 comprising sources of carbon, nitrogen, andnutrients, and about 950 ppm to about 8500 ppm chloride ions.

In some embodiments, the fermentation volume (volume of culture) is atleast about 2 liters, at least about 10 liters, at least about 50liters, at least about 100 liters, at least about 200 liters, at leastabout 500 liters, at least about 1000 liters, at least about 10,000liters, at least about 20,000 liters, at least about 50,000 liters, atleast about 100,000 liters, at least about 150,000 liters, at leastabout 200,000 liters, or at least about 250,000 liters. In someembodiments, the fermentation volume is about 2 liters to about 300,000liters, about 2 liters, about 10 liters, about 50 liters, about 100liters, about 200 liters, about 500 liters, about 1000 liters, about10,000 liters, about 20,000 liters, about 50,000 liters, about 100,000liters, about 150,000 liters, about 200,000 liters, about 250,000liters, or about 300,000 liters.

In some embodiments, the invention is directed to an isolatedthraustochytrid biomass comprising a fatty acid profile of theinvention. In some embodiments, at least about 50%, at least about 60%,at least about 70%, or at least about 80% of the dry cell weight of thebiomass are fatty acids. In some embodiments, greater than about 50%,greater than about 55%, or greater than about 60% of the dry cell weightof the biomass are fatty acids. In some embodiments, from about 50% toabout 60%, about 50% to about 70%, about 50% to about 80%, about 55% toabout 70%, about 55% to about 80%, about 60% to about 70%, or about 60%to about 80% by weight of the dry cell weight of the biomass are fattyacids. In some embodiments, the biomass comprises at least about 50%, atleast about 60%, at least about 70%, or at least about 80% by weight ofthe fatty acids as omega-3 fatty acids. In some embodiments, the biomasscomprises from about 50% to about 60%, about 50% to about 70%, about 50%to about 80% by weight of the fatty acids as omega-3 fatty acids. Insome embodiments, the biomass comprises at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, or at least about 80% by weight of the fatty acidsas DHA. In some embodiments, the biomass comprises from about 50% toabout 60%, about 50% to about 70%, or about 50% to about 80% by weightof the fatty acids as DHA. In some embodiments, at least about 25%, atleast about 30%, at least about 40%, at least about 50%, or at leastabout 60% by weight of the dry cell weight of the biomass isdocosahexaenoic acid. In some embodiments, about 25% to about 65%, about25% to about 50%, about 30% to about 40%, or about 25% to about 35% byweight of the dry cell weight of the biomass is docosahexaenoic acid. Insome embodiments, the biomass comprises about 10% or less, about 9% orless, about 8% or less, about 7% or less, about 6% or less, about 5% orless, about 4% or less, about 3% or less, about 2% or less, or about 1%or less by weight of the fatty acids as EPA. In some embodiments, thebiomass comprises from about 1% to about 10%, about 1% to about 5%,about 2% to about 5%, about 3% to about 5%, or about 3% to about 10% byweight of the fatty acids as EPA. In some embodiments, the biomass issubstantially free of EPA. In some embodiments, the biomass comprises aweight ratio of DHA to EPA of at least about 5:1, at least about 7:1, atleast about 10:1, at least about 11:1, at least about 14:1, at leastabout 15:1, at least about 17:1, at least about 20:1, at least about25:1, at least about 50:1, or at least about 100:1, wherein the biomasscomprises about 10% or less by weight of the fatty acids as EPA. In someembodiments, the biomass comprises from about 0.1% to 0.2%, about 0.1%to about 0.3%, about 0.1% to about 0.4%, about 0.1% to about 0.5%, orabout 0.1% to about 1.5% by weight of the fatty acids as ARA. In someembodiments, the biomass comprises about 1.5% or less, about 1% or less,about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2%or less, or about 0.1% or less by weight of the fatty acids as ARA. Insome embodiments, the biomass is substantially free of ARA. In someembodiments, the biomass comprises a weight ratio of DHA to ARA of atleast about 20:1, at least about 40:1, at least about 60:1, at leastabout 80:1, at least about 100:1, at least about 150:1, at least about200:1, at least about 250:1, or at least about 300:1. In someembodiments, the biomass comprises from about 0.5% to about 1%, about0.5% to about 2%, about 0.5% to about 5%, about 0.5% to about 6%, about1% to about 5%, about 1% to about 6%, about 2% to about 5%, or about 2%to about 6% by weight of the fatty acids as DPA n-6. In someembodiments, the biomass comprises about 6% or less, about 5% or less,about 2% or less, about 1% or less, or about 0.5% or less by weight ofthe fatty acids as DPA n-6. In some embodiments, the biomass issubstantially free of DPA n-6. In some embodiments, the biomasscomprises a weight ratio of DHA to DPA n-6 of greater than about 6:1, atleast about 8:1, at least about 10:1, at least about 15:1, at leastabout 20:1, at least about 25:1, at least about 50:1, or at least about100:1. In some embodiments, the biomass comprises fatty acids with about5% or less, about 4% or less, about 3% or less, or about 2% or less byweight each of linoleic acid (18:2 n-6), linolenic acid (18:3 n-3),eicosenoic acid (20:1 n-9), and erucic acid (22:1 n-9).

The characteristics of an isolated biomass of the invention areassociated with endogenous or native properties of the isolated biomassrather than exogenously introduced materials.

Microbial Oils

The present invention is further directed to methods of producingmicrobial oils.

In some embodiments, the method comprises growing a thraustochytrid ofthe invention in a culture to produce a biomass and extracting an oilcomprising omega-3 fatty acids from the biomass. The oil can beextracted from a freshly harvested biomass or can be extracted from apreviously harvested biomass that has been stored under conditions thatprevent spoilage. Known methods can be used to culture a thraustochytridof the invention, to isolate a biomass from the culture, to extract amicrobial oil from the biomass, and to analyze the fatty acid profile ofoils extracted from the biomass. See, e.g., U.S. Pat. No. 5,130,242.

The invention is further directed to a microbial oil comprising a fattyacid profile of the invention. A microbial oil of the invention can beany oil derived from a microorganism, including, for example: a crudeoil extracted from the biomass of the microorganism without furtherprocessing; a refined oil that is obtained by treating a crude microbialoil with further processing steps such as refining, bleaching, and/ordeodorizing; a diluted microbial oil obtained by diluting a crude orrefined microbial oil; or an enriched oil that is obtained, for example,by treating a crude or refined microbial oil with further methods ofpurification to increase the concentration of a fatty acid (such as DHA)in the oil.

In some embodiments, the microbial oil comprises a sterol estersfraction of about 0%, at least about 0.1%, at least about 0.2%, at leastabout 0.5%, at least about 1%, at least about 1.5%, at least about 2%,or at least about 5% by weight. In some embodiments, the microbial oilcomprises a sterol esters fraction of from about 0% to about 1.5%, about0% to about 2%, about 0% to about 5%, about 1% to about 1.5%, about 0.2%to about 1.5%, about 0.2% to about 2%, or about 0.2% to about 5% byweight. In some embodiments, the microbial oil comprises a sterol estersfraction of less than about 5%, less than about 4%, less than about 3%,or less than about 2% by weight. In some embodiments, the microbial oilcomprises a triglyceride fraction of at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, or atleast about 90% by weight. In some embodiments, the microbial oilcomprises a triglyceride fraction of from about 65% to about 95%, about75% to about 95%, or about 80% to about 95% by weight, or about 97% byweight, or about 98% by weight. In some embodiments, the microbial oilcomprises a free fatty acid fraction of at least about 0.5%, at leastabout 1%, at least about 1.5%, at least about 2%, at least about 2.5%,or at least about 5% by weight. In some embodiments, the microbial oilcomprises a free fatty acid fraction of from about 0.5% to about 5%,about 0.5% to about 2.5%, about 0.5% to about 2%, about 0.5% to about1.5%, about 0.5% to about 1%, about 1% to about 2.5%, about 1% to about5%, about 1.5% to about 2.5%, about 2% to about 2.5%, or about 2% toabout 5% by weight. In some embodiments, the microbial oil comprises afree fatty acid fraction of less than about 5%, less than about 4%, lessthan about 3%, less than about 2%, or less than about 1% by weight. Insome embodiments, the microbial oil comprises a sterol fraction of atleast about 0.5%, at least about 1%, at least about 1.5%, at least about2%, or at least about 5% by weight. In some embodiments, the microbialoil comprises a sterol fraction of from about 0.5% to about 1.5%, about1% to about 1.5%, about 0.5% to about 2%, about 0.5% to about 5%, about1% to about 2%, or about 1% to about 5% by weight. In some embodiments,the microbial oil comprises a sterol fraction of less than about 5%,less than about 4%, less than about 3%, less than about 2%, or less thanabout 1% by weight. In some embodiments, the microbial oil comprises adiglyceride fraction of at least about 1.5%, at least about 2%, at leastabout 2.5%, at least about 3%, at least about 3.5%, or at least about 5%by weight. In some embodiments, the microbial oil comprises adiglyceride fraction of from about 1.5% to about 3%, about 2% to about3%, about 1.5% to about 3.5%, about 1.5% to about 5%, about 2.5% toabout 3%, about 2.5% to about 3.5%, or about 2.5% to about 5% by weight.In some embodiments, the microbial oil comprises unsaponifiables of lessthan about 2%, less than about 1.5%, less than about 1%, or less thanabout 0.5% by weight of the oil. The lipid classes present in themicrobial oil, such as the triglyceride fraction, can be separated byflash chromatography and analyzed by thin layer chromatography (TLC), orseparated and analyzed by other methods know in the art.

In some embodiments, the microbial oil and/or one or more fractionsthereof selected from the triglyceride fraction, the free fatty acidfraction, the sterol fraction, the diglyceride fraction, andcombinations thereof, comprises at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, or at least about 80%by weight DHA. In some embodiments, the microbial oil and/or one or morefractions thereof selected from the triglyceride fraction, the freefatty acid fraction, the sterol fraction, the diglyceride fraction, andcombinations thereof, comprises from about 40% to about 45%, about 40%to about 50%, about 40% to about 60%, about 50% to about 60%, about 55%to about 60%, about 40% to about 65%, about 50% to about 65%, about 55%to about 65%, about 40% to about 70%, about 40% to about 80%, about 50%to about 80%, about 55% to about 80%, about 60% to about 80%, or about70% to about 80% by weight DHA. In some embodiments, the microbial oilcomprises a sterol esters fraction comprising about 45% or less, about40% or less, about 35% or less, about 30% or less, about 25% or less,about 20% or less, about 15% or less, or about 13% or less by weightDHA. In some embodiments, the microbial oil and/or one or more fractionsthereof selected from the triglyceride fraction, the free fatty acidfraction, the sterol fraction, the diglyceride fraction, andcombinations thereof, comprises about 10% or less, about 9% or less,about 8% or less, about 7% or less, about 6% or less, about 5% or less,about 4% or less, about 3% or less, about 2% or less, or about 1% orless by weight EPA. In some embodiments, the microbial oil and/or one ormore fractions thereof selected from the triglyceride fraction, the freefatty acid fraction, the sterol fraction, the diglyceride fraction, andcombinations thereof, comprises from about 2% to about 3%, about 2% toabout 3.5%, about 2.5% to about 3.5%, about 2% to about 6%, about 2.5%to about 6%, about 3.0% to about 6%, about 3.5% to about 6%, about 5% toabout 6%, or about 2% to about 10% by weight EPA. In some embodiments,the microbial oil and/or one or more fractions thereof selected from thesterol esters fraction, the triglyceride fraction, the free fatty acidfraction, the sterol fraction, the diglyceride fraction, the polarfraction (including the phospholipid fraction), and combinationsthereof, is substantially free of EPA. In some embodiments, themicrobial oil and/or one or more fractions thereof selected from thesterol esters fraction, the triglyceride fraction, the free fatty acidfraction, the sterol fraction, the diglyceride fraction, the polarfraction (including the phospholipid fraction), and combinationsthereof, comprises a weight ratio of DHA to EPA of at least about 5:1,at least about 7:1, at least about 9:1, at least about 10:1, at leastabout 15:1, at least about 20:1, at least about 25:1, at least about30:1, or at least about 50:1, wherein the microbial oil and/or one ormore fractions thereof comprises 10% or less by weight of EPA. In someembodiments, the microbial oil and/or one or more fractions thereofselected from the sterol esters fraction, the triglyceride fraction, thefree fatty acid fraction, the sterol fraction, the diglyceride fraction,the polar fraction (including the phospholipid fraction), andcombinations thereof, comprises a weight ratio of DHA to EPA of at leastabout 5:1, but less than about 20:1. In some embodiments, the weightratio of DHA to EPA is from about 5:1 to about 18:1, from about 7:1 toabout 16:1, or from about 10:1 to about 15:1. In some embodiments, themicrobial oil and/or one or more fractions thereof selected from thesterol esters fraction, the triglyceride fraction, the free fatty acidfraction, the sterol fraction, the diglyceride fraction, the polarfraction (including the phospholipid fraction), and combinations thereofcomprises from about 0.1% to about 0.25%, about 0.2% to about 0.25%,about 0.1% to about 0.5%, or about 0.1% to about 1.5% by weight ARA. Insome embodiments, the microbial oil and/or one or more fractions thereofselected from the sterol esters fraction, the triglyceride fraction, thefree fatty acid fraction, the sterol fraction, the diglyceride fraction,the polar fraction (including the phospholipid fraction), andcombinations thereof, comprises about 1.5% or less, about 1% or less,about 0.5% or less, about 0.2% or less, or about 0.1% or less by weightARA. In some embodiments, the microbial oil and/or one or more fractionsthereof selected from the sterol esters fraction, the triglyceridefraction, the free fatty acid fraction, the sterol fraction, thediglyceride fraction, the polar fraction (including the phospholipidfraction), and combinations thereof, is substantially free of ARA. Insome embodiments, the microbial oil and/or one or more fractions thereofselected from the sterol esters fraction, the triglyceride fraction, thefree fatty acid fraction, the diglyceride fraction, the polar fraction(including the phospholipid fraction), and combinations thereof,comprises a weight ratio of DHA to ARA of at least about 20:1, at leastabout 30:1, at least about 35:1, at least about 40:1, at least about60:1, at least about 80:1, at least about 100:1, at least about 150:1,at least about 200:1, at least about 250:1, or at least about 300:1. Insome embodiments, the microbial oil and/or one or more fractions thereofselected from the sterol esters fraction, the triglyceride fraction, thefree fatty acid fraction, the sterol fraction, the diglyceride fraction,the polar fraction (including the phospholipid fraction), andcombinations thereof, comprises from about 0.5% to about 1%, about 0.5%to about 2%, about 0.5% to about 2.5%, about 0.5% to about 3%, about0.5% to about 3.5%, about 0.5% to about 5%, about 0.5% to about 6%,about 1% to about 2%, about 2% to about 3%, about 2% to about 3.5%,about 1% to about 2.5%, about 1% to about 3%, about 1% to about 3.5%,about 1% to about 5%, or about 1% to about 6% by weight DPA n-6. In someembodiments, the microbial oil and/or one or more fractions thereofselected from the sterol esters fraction, the triglyceride fraction, thefree fatty acid fraction, the sterol fraction, the diglyceride fraction,the polar fraction (including the phospholipid fraction), andcombinations thereof, comprises about 6% or less, about 5% or less,about 3% or less, about 2.5% or less, about 2% or less, about 1% orless, or about 0.5% or less by weight DPA n-6. In some embodiments, themicrobial oil and/or one or more fractions thereof selected from thesterol esters fraction, the triglyceride fraction, the free fatty acidfraction, the sterol fraction, the diglyceride fraction, the polarfraction (including the phospholipid fraction), and combinationsthereof, is substantially free of DPA n-6. In some embodiments, themicrobial oil and/or one or more fractions thereof selected from thesterol esters fraction, the triglyceride fraction, the free fatty acidfraction, the sterol fraction, the diglyceride fraction, the polarfraction (including the phospholipid fraction), and combinationsthereof, comprises a weight ratio of DHA to DPA n-6 of greater thanabout 6:1, of at least about 8:1, at least about 10:1, at least about15:1, at least about 20:1, at least about 25:1, at least about 50:1, orat least about 100:1. In some embodiments, the microbial oil and/or oneor more fractions thereof selected from the sterol esters fraction, thetriglyceride fraction, the free fatty acid fraction, the sterolfraction, the diglyceride fraction, the polar fraction (including thephospholipid fraction), and combinations thereof, comprises about 5% orless, about 4% or less, about 3% or less, about 2% or less, about 1.5%or less, about 1% or less, or about 0.5% or less by weight each oflinoleic acid (18:2 n-6), linolenic acid (18:3 n-3), eicosenoic acid(20:1 n-9), and erucic acid (22:1 n-9). In some embodiments, themicrobial oil and/or one or more fractions thereof selected from thesterol esters fraction, the triglyceride fraction, the free fatty acidfraction, the sterol fraction, the diglyceride fraction, the polarfraction (including the phospholipid fraction), and combinationsthereof, comprises about 5% or less, about 4% or less, about 3% or less,about 2% or less, about 1.5% or less, or about 1% or less by weight ofheptadecanoic acid (17:0). In some embodiments, the microbial oil and/orone or more fractions thereof comprise about 0.01% to about 5% byweight, about 0.05% to about 3% by weight, or about 0.1% to about 1% byweight of heptadecanoic acid.

The triglyceride molecule contains 3 central carbon atoms(C_(sn-1)H₂R1-C_(sn-2)H₂R2-C_(sn-3)H₂R3), allowing for formation ofdifferent positional isomers. In some embodiments, the microbial oilcomprises a triglyceride fraction in which at least about 20%, at leastabout 30%, at least about 35%, or at least about 40% of thetriglycerides in the triglyceride fraction contain DHA at two positionsin the triglyceride (di-substituted DHA) selected from any two of thesn-1, sn-2, and sn-3 positions, based on the relative area percent ofpeaks on an HPLC chromatograph. In some embodiments, the microbial oilcomprises a triglyceride fraction in which from about 20% to about 40%,about 20% to about 35%, about 30% to about 40%, or about 30% to about35% of the triglycerides in the triglyceride fraction contain DHA at twopositions in the triglyceride selected from any two of the sn-1, sn-2,or sn-3 positions, based on the relative area percent of peaks on anHPLC chromatograph. In some embodiments, the microbial oil comprises atriglyceride fraction in which at least about 5%, at least about 10%, atleast about 15%, or at least about 20% of the triglycerides in thetriglyceride fraction contain DHA at all of the sn-1, sn-2, and sn-3positions (tri-substituted DHA), based on the relative area percent ofpeaks on an HPLC chromatograph. In some embodiments, the microbial oilcomprises a triglyceride fraction in which from about 5% to about 20%,about 5% to about 15%, about 10% to about 20%, or about 10% to about 15%of the triglycerides in the triglyceride fraction contain DHA at all ofthe sn-1, sn-2, and sn-3 positions, based on the relative area percentof peaks on an HPLC chromatograph. In contrast, the TAG species reportedin U.S. Pat. No. 6,582,941 does not contain DHA at all three positions.In some embodiments, the microbial oil comprises a triglyceride fractionin which at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, or at least about 75% of thetriglycerides in the triglyceride fraction contain DHA at one positionin the triglyceride selected from any one of the sn-1, sn-2, or sn-3positions, based on the relative area percent of peaks on an HPLCchromatograph. In some embodiments, the microbial oil comprises atriglyceride fraction in which from about 50% to about 75%, about 50% toabout 70%, about 50% to about 65%, about 60% to about 75%, about 60% toabout 70%, or about 60% to about 65% of the triglycerides in thetriglyceride fraction contain DHA at one position in the triglycerideselected from any one of the sn-1, sn-2, and sn-3 positions, based onthe relative area percent of peaks on an HPLC chromatograph.

Compositions

The invention is further directed to compositions comprising athraustochytrid of the invention, an isolated biomass of the invention,a microbial oil of the invention, or combinations thereof.

A thraustochytrid, biomass, or microbial oil of the invention can befurther chemically or physically modified or processed based on therequirements of the composition by any known technique.

Thraustochytrid cells or biomasses can be dried prior to use in acomposition by methods including, but not limited to, freeze drying, airdrying, spray drying, tunnel drying, vacuum drying (lyophilization), ora similar process. Alternatively, a harvested and washed biomass can beused directly in a composition without drying. See, e.g., U.S. Pat. Nos.5,130,242 and 6,812,009.

Microbial oils of the invention can be used as starting material to moreefficiently produce a product enriched in a fatty acid such as DHA. Forexample, the microbial oils of the invention can be subjected to variouspurification techniques known in the art, such as distillation or ureaadduction, to produce a higher potency product with higherconcentrations of DHA or another fatty acid. The microbial oils of theinvention can also be used in chemical reactions to produce compoundsderived from fatty acids in the oils, such as esters and salts of DHA oranother fatty acid.

A composition of the invention can include one or more excipients. Asused herein, “excipient” refers to a component, or mixture ofcomponents, that is used in a composition of the present invention togive desirable characteristics to the composition, including foods aswell as pharmaceutical, cosmetic, and industrial compositions. Anexcipient of the present invention can be described as a“pharmaceutically acceptable” excipient when added to a pharmaceuticalcomposition, meaning that the excipient is a compound, material,composition, salt, and/or dosage form which is, within the scope ofsound medical judgment, suitable for contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problematic complications over the desired durationof contact commensurate with a reasonable benefit/risk ratio. In someembodiments, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized internationalpharmacopeia for use in animals, and more particularly in humans.Various excipients can be used. In some embodiments, the excipient canbe, but is not limited to, an alkaline agent, a stabilizer, anantioxidant, an adhesion agent, a separating agent, a coating agent, anexterior phase component, a controlled-release component, a solvent, asurfactant, a humectant, a buffering agent, a filler, an emollient, orcombinations thereof. Excipients in addition to those discussed hereincan include excipients listed in, though not limited to, Remington: TheScience and Practice of Pharmacy, 21^(st) ed. (2005). Inclusion of anexcipient in a particular classification herein (e.g., “solvent”) isintended to illustrate rather than limit the role of the excipient. Aparticular excipient can fall within multiple classifications.

Compositions of the invention include, but are not limited to, foodproducts, pharmaceutical compositions, cosmetics, and industrialcompositions.

In some embodiments, the composition is a food product. A food productis any food for animal or human consumption, and includes both solid andliquid compositions. A food product can be an additive to animal orhuman foods. Foods include, but are not limited to, common foods; liquidproducts, including milks, beverages, therapeutic drinks, andnutritional drinks; functional foods; supplements; nutraceuticals;infant formulas, including formulas for pre-mature infants; foods forpregnant or nursing women; foods for adults; geriatric foods; and animalfoods.

In some embodiments, a thraustochytrid, biomass, or microbial oil of theinvention can be used directly as or included as an additive within oneor more of: an oil, shortening, spread, other fatty ingredient,beverage, sauce, dairy-based or soy-based food (such as milk, yogurt,cheese and ice-cream), a baked good, a nutritional product, e.g., as anutritional supplement (in capsule or tablet form), a vitaminsupplement, a diet supplement, a powdered drink, a finished orsemi-finished powdered food product, and combinations thereof.

A partial list of food compositions that can include a microbial oil ofthe invention includes, but is not limited to, soya based products(milks, ice creams, yogurts, drinks, creams, spreads, whiteners); soupsand soup mixes; doughs, batters, and baked food items including, forexample, fine bakery wares, breakfast cereals, cakes, cheesecakes, pies,cupcakes, cookies, bars, breads, rolls, biscuits, muffins, pastries,scones, croutons, crackers, sweet goods, snack cakes, pies,granola/snack bars, and toaster pastries; candy; hard confectionery;chocolate and other confectionery; chewing gum; liquid food products,for example milks, energy drinks, infant formula, carbonated drinks,teas, liquid meals, fruit juices, fruit-based drinks, vegetable-baseddrinks; multivitamin syrups, meal replacers, medicinal foods, andsyrups; powdered beverage mixes; pasta; processed fish products;processed meat products; processed poultry products; gravies and sauces;condiments (ketchup, mayonnaise, etc.); vegetable oil-based spreads;dairy products; yogurt; butters; frozen dairy products; ice creams;frozen desserts; frozen yogurts; semi-solid food products such as babyfood; puddings and gelatin desserts; processed and unprocessed cheese;pancake mixes; food bars including energy bars; waffle mixes; saladdressings; replacement egg mixes; nut and nut-based spreads; saltedsnacks such as potato chips and other chips or crisps, corn chips,tortilla chips, extruded snacks, popcorn, pretzels, potato crisps, andnuts; specialty snacks such as dips, dried fruit snacks, meat snacks,pork rinds, health food bars and rice/corn cakes.

In some embodiments, a microbial oil of the invention can be used tosupplement infant formula. Infant formula can be supplemented with amicrobial oil of the invention alone or in combination with a physicallyrefined oil derived from an arachidonic acid (ARA)-producingmicroorganism. An ARA-producing microorganism, for example, isMortierella alpina or Mortierella sect. schmuckeri. Alternatively,infant formulas can be supplemented with a microbial oil of theinvention in combination with an oil rich in ARA, including ARASCO®(Martek Biosciences, Columbia, Md.).

In some embodiments, the composition is an animal feed. An “animal”means any non-human organism belonging to the kingdom Animalia, andincludes, without limitation, aquatic animals and terrestrial animals.The term “animal feed” or “animal food” refers to any food intended fornon-human animals, whether for fish; commercial fish; ornamental fish;fish larvae; bivalves; mollusks; crustaceans; shellfish; shrimp; larvalshrimp; artemia; rotifers; brine shrimp; filter feeders; amphibians;reptiles; mammals; domestic animals; farm animals; zoo animals; sportanimals; breeding stock; racing animals; show animals; heirloom animals;rare or endangered animals; companion animals; pet animals such as dogs,cats, guinea pigs, rabbits, rats, mice, or horses; primates such asmonkeys (e.g., cebus, rhesus, African green, patas, cynomolgus, andcercopithecus), apes, orangutans, baboons, gibbons, and chimpanzees;canids such as dogs and wolves; felids such as cats, lions, and tigers;equids such as horses, donkeys, and zebras; food animals such as cows,cattle, pigs, and sheep; ungulates such as deer and giraffes; rodentssuch as mice, rats, hamsters and guinea pigs; and so on. An animal feedincludes, but is not limited to, an aquaculture feed, a domestic animalfeed including pet feed, a zoological animal feed, a work animal feed, alivestock feed, or a combination thereof.

In some embodiments, the composition is a feed or feed supplement forany animal whose meat or products are consumed by humans, such as anyanimal from which meat, eggs, or milk is derived for human consumption.When fed to such animals, nutrients such as LC-PUFAs can be incorporatedinto the flesh, milk, eggs or other products of such animals to increasetheir content of these nutrients.

In some embodiments, the composition is a spray-dried material that canbe crumbled to form particles of an appropriate size for consumption byzooplankton, artemia, rotifers, and filter feeders. In some embodiments,the zooplankton, artemia, or rotifers fed by the composition are in turnfed to fish larvae, fish, shellfish, bivalves, or crustaceans.

In some embodiments, the composition is a pharmaceutical composition.Suitable pharmaceutical compositions include, but are not limited to, ananti-inflammatory composition, a drug for treatment of coronary heartdisease, a drug for treatment of arteriosclerosis, a chemotherapeuticagent, an active excipient, an osteoporosis drug, an anti-depressant, ananti-convulsant, an anti-Helicobacter pylori drug, a drug for treatmentof neurodegenerative disease, a drug for treatment of degenerative liverdisease, an antibiotic, a cholesterol lowering composition, and atriglyceride lowering composition. In some embodiments, the compositionis a medical food. A medical food includes a food that is in acomposition to be consumed or administered externally under thesupervision of a physician and that is intended for the specific dietarymanagement of a condition, for which distinctive nutritionalrequirements, based on recognized scientific principles, are establishedby medical evaluation.

In some embodiments, the microbial oil can be formulated in a dosageform. Dosage forms can include, but are not limited to, tablets,capsules, cachets, pellets, pills, powders and granules, and parenteraldosage forms, which include, but are not limited to, solutions,suspensions, emulsions, and dry powders comprising an effective amountof the microbial oil. It is also known in the art that such formulationscan also contain pharmaceutically acceptable diluents, fillers,disintegrants, binders, lubricants, surfactants, hydrophobic vehicles,water soluble vehicles, emulsifiers, buffers, humectants, moisturizers,solubilizers, preservatives and the like. Administration forms caninclude, but are not limited to, tablets, dragees, capsules, caplets,and pills, which contain the microbial oil and one or more suitablepharmaceutically acceptable carriers.

For oral administration, the microbial oil can be combined withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the microbial oils of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.In some embodiments, the dosage form is a tablet, pill or caplet.Pharmaceutical preparations for oral use can be obtained by adding asolid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired, to obtain tablets or dragee cores. Suitable excipientsinclude, but are not limited to, fillers such as sugars, including, butnot limited to, lactose, sucrose, mannitol, and sorbitol; cellulosepreparations such as, but not limited to, maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, andpolyvinylpyrrolidone (PVP). If desired, disintegrating agents can beadded, such as, but not limited to, the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. Pharmaceutical preparations that can be used orally include,but are not limited to, push-fit capsules made of gelatin, as well assoft, sealed capsules made of gelatin and a plasticizer, such asglycerol or sorbitol.

In some embodiments, the composition is a cosmetic. Cosmetics include,but are not limited to, emulsions, creams, lotions, masks, soaps,shampoos, washes, facial creams, conditioners, make-ups, bath agents,and dispersion liquids. Cosmetic agents can be medicinal ornon-medicinal.

In some embodiments, the composition is an industrial composition. Insome embodiments, the composition is a starting material for one or moremanufactures. A manufacture includes, but is not limited to, a polymer;a photographic photosensitive material; a detergent; an industrial oil;or an industrial detergent. For example, U.S. Pat. No. 7,259,006describes use of DHA-containing fat and oil for production of behenicacid and production of photographic sensitive materials using behenicacid.

Methods of Using the Compositions

In some embodiments, the compositions can be used in the treatment of acondition in humans or animals.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological condition,disease, or disorder, or to obtain beneficial or desired clinicalresults. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of the symptoms orsigns associated with a condition, disease, or disorder; diminishment ofthe extent of a condition, disease, or disorder; stabilization of acondition, disease, or disorder, (i.e., where the condition, disease, ordisorder is not worsening); delay in onset or progression of thecondition, disease, or disorder; amelioration of the condition, disease,or disorder; remission (whether partial or total and whether detectableor undetectable) of the condition, disease, or disorder; or enhancementor improvement of a condition, disease, or disorder. Treatment includeseliciting a clinically significant response without excessive sideeffects. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment.

In some embodiments, the composition is used to treat a condition,disease, or disorder such as acne, acute inflammation, age relatedmaculopathy, allergy, Alzheimer's, arthritis, asthma, atherosclerosis,autoimmune disease, blood lipid disorder, breast cysts, cachexia,cancer, cardiac restenosis, cardiovascular diseases, chronicinflammation, coronary heart disease, cystic fibrosis, degenerativedisorder of the liver, diabetes, eczema, gastrointestinal disorder,heart disease, high triglyceride levels, hypertension, hyperactivity,immunological diseases, inhibiting tumor growth, inflammatoryconditions, intestinal disorders, kidney dysfunction, leukemia, majordepression, multiple sclerosis, neurodegenerative disorder,osteoarthritis, osteoporosis, peroxisomal disorder, preeclampsia,preterm birth, psoriasis, pulmonary disorder rheumatoid arthritis, riskof heart disease, or thrombosis.

In some embodiments, the composition is used to increase the length ofgestation in the third trimester.

In some embodiments, the composition is used to control blood pressure.

In some embodiments, the composition is used to improve or maintaincognitive function.

In some embodiments, the composition is used to improve or maintainmemory.

The composition or dosage form can be administered into the body of asubject by any route compatible with the composition or dosage form. Asubstance is considered to be “administered” if the substance isintroduced into the body of the subject by the subject, or if anotherperson, a machine, or a device introduces the substance into the body ofthe subject. “Administering,” therefore, includes, e.g.,self-administration, administration by others, and indirectadministration. The term “continuous” or “consecutive,” as used hereinin reference to “administration,” means that the frequency ofadministration is at least once daily. Note, however, that the frequencyof administration can be greater than once daily and still be“continuous” or “consecutive,” e.g., twice or even three times daily, aslong as the dosage levels as specified herein are not exceeded. Themeans and methods for administration are known in the art and an artisancan refer to various pharmacologic references for guidance. For example,“Modern Pharmaceutics,” Banker & Rhodes, Informa Healthcare, USA, 4thed. (2002); and “Goodman & Gilman's The Pharmaceutical Basis ofTherapeutics,” McGraw-Hill Companies, Inc., New York, 10th ed. (2001)can be consulted.

By “subject,” “individual,” or “patient” is meant any subject, whetherhuman or non-human, for whom diagnosis, prognosis, or therapy isdesired. Mammalian subjects include, but are not limited to, humans;domestic animals; farm animals; zoo animals; sport animals; pet animalssuch as dogs, cats, guinea pigs, rabbits, rats, mice, or horses;primates such as monkeys (e.g., cebus, rhesus, African green, patas,cynomolgus, and cercopithecus), apes, orangutans, baboons, gibbons, andchimpanzees; canids such as dogs and wolves; felids such as cats, lions,and tigers; equids such as horses, donkeys, and zebras; food animalssuch as cows, cattle, pigs, and sheep; ungulates such as deer andgiraffes; rodents such as mice, rats, hamsters and guinea pigs; and soon. The term subject also encompasses model animals, e.g., disease modelanimals. In some embodiments, the term subject includes valuableanimals, either economically or otherwise, e.g., economically importantbreeding stock, racing animals, show animals, heirloom animals, rare orendangered animals, or companion animals. In certain embodiments, thesubject is a human subject. In certain embodiments, the subject is anon-human subject.

The composition can be administered as a “therapeutically effectiveamount,” a “prophylactically effective amount,” a “therapeutic dose,” ora “prophylactic dose.” A “therapeutically effective amount” or“therapeutic dose” refers to an amount effective, at dosages and forperiods of time necessary, to achieve a desired therapeutic result. Atherapeutic result can be, e.g., lessening of symptoms, prolongedsurvival, improved mobility, and the like. A therapeutic result need notbe a “cure.” A “prophylactically effective amount” or “prophylacticdose” refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired prophylactic result. Typically, sincea prophylactic dose is used in subjects prior to or at an earlier stageof disease, a prophylactically effective amount will be less than atherapeutically effective amount for treatment of an advanced stage ofdisease.

Various dosage amounts of the pharmaceutical composition can beadministered to a subject, based on the amount of DHA or other fattyacid component of the thraustochytrid, biomass, or microbial oil to beadministered to the subject. The terms “daily dosage,” “daily dosagelevel,” and “daily dosage amount” refer herein to the total amount ofDHA or other fatty acid component administered per day (per 24 hourperiod). Thus, for example, administration of DHA to a subject at adaily dosage of 2 mg means that the subject receives a total of 2 mg ofDHA on a daily basis, whether the DHA is administered as a single dosageform comprising 2 mg DHA, or alternatively, four dosage forms comprising0.5 mg DHA each (for a total of 2 mg DHA). In some embodiments, thedaily amount of DHA is administered in a single dosage form, or in twodosage forms. The dosage forms of the present invention can be taken ina single application or multiple applications. For example, if fourtablets are taken daily, each tablet comprising 0.5 mg DHA, then allfour tablets can be taken once daily, or 2 tablets can be taken twicedaily, or 1 tablet can be taken every 6 hours. In some embodiments, thedaily dosage is from about 100 mg to about 15 g of DHA. In someembodiments, the daily dosage is from about 100 mg to about 250 mg,about 100 mg to about 500 mg, about 100 mg to about 1 g, about 1 g toabout 2.5 g, about 1 g to about 5 g, about 1 g to about 10 g, about 1 gto about 15 g, about 5 g to about 10 g, about 5 g to about 15 g, about10 g to about 15 g, about 100 mg to about 10 g, about 100 mg to about 5g, or about 100 mg to about 2.5 g.

Administration of the compositions or dosage forms of the presentinvention can be achieved using various regimens. For example, in someembodiments, administration occurs daily on consecutive days, oralternatively, occurs every other day (bi-daily). Administration canoccur on one or more days.

Administration of the compositions and dosage forms can be combined withother regimens used for treatment of the condition. For example, themethod of the present invention can be combined with diet regimens(e.g., low carbohydrate diets, high protein diets, high fiber diets,etc.), exercise regimens, weight loss regimens, smoking cessationregimens, or combinations thereof. The method of the present inventioncan also be used in combination with other pharmaceutical products inthe treatment of the condition. The compositions or dosage forms of thepresent invention can be administered before or after other regimens orpharmaceutical products.

Kits Comprising the Compositions

The invention is further directed to kits or packages containing one ormore units of a composition of the invention. Kits or packages caninclude units of a food product, pharmaceutical composition, cosmetic,or industrial composition comprising the thraustochytrid, biomass, ormicrobial oil of the invention, or combinations thereof. Kits orpackages can also include an additive comprising the thraustochytrid,biomass, or microbial oil of the invention, or combinations thereof forpreparation of a food, cosmetic, pharmaceutical composition, orindustrial composition.

In some embodiments, the kit or package contains one or more units of apharmaceutical composition to be administered according to the methodsof the present invention. The kit or package can contain one dosageunit, or more than one dosage unit (i.e., multiple dosage units). Ifmultiple dosage units are present in the kit or package, the multipledosage units can be optionally arranged for sequential administration.

The kits of the present invention can optionally contain instructionsassociated with the units or dosage forms of the kits. Such instructionscan be in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceutical products, which noticereflects approval by the agency of the manufacture, use or sale forhuman administration to treat a condition or disorder. The instructionscan be in any form which conveys information on the use of the units ordosage forms in the kit according to the methods of the invention. Forexample, the instructions can be in the form of printed matter, or inthe form of a pre-recorded media device.

In the course of examination of a patient, a medical professional candetermine that administration of one of the methods of the presentinvention is appropriate for the patient, or the physician can determinethat the patient's condition can be improved by the administration ofone of the methods of the present invention. Prior to prescribing anyregimen, the physician can counsel the patient, for example, on thevarious risks and benefits associated with the regimen. The patient canbe provided full disclosure of all known and suspected risks associatedwith the regimen. Such counseling can be provided verbally, as well asin written form. In some embodiments, the physician can provide thepatient with literature materials on the regimen, such as productinformation, educational materials, and the like.

The present invention is also directed to methods of educating consumersabout the methods of treatment, the method comprising distributing thedosage forms with consumer information at a point of sale. In someembodiments, the distribution will occur at a point of sale having apharmacist or healthcare provider.

The term “consumer information” can include, but is not limited to, anEnglish language text, non-English language text, visual image, chart,telephone recording, website, and access to a live customer servicerepresentative. In some embodiments, consumer information will providedirections for use of the dosage forms according to the methods of thepresent invention, appropriate age use, indication, contraindications,appropriate dosing, warnings, telephone number of website address. Insome embodiments, the method further comprises providing professionalinformation to relevant persons in a position to answer consumerquestions regarding use of the disclosed regimens according to themethods of the present invention. The term “professional information”includes, but is not limited to, information concerning the regimen whenadministered according to the methods of the present invention that isdesigned to enable a medical professional to answer customer questions.

A “medical professional,” includes, for example, a physician, physicianassistant, nurse practitioner, pharmacist and customer servicerepresentative.

Having generally described this invention, a further understanding canbe obtained by reference to the examples provided herein. These examplesare for purposes of illustration only and are not intended to belimiting.

EXAMPLE 1

The isolated thraustochytrid deposited under ATCC Accession No. PTA-9695was characterized for taxonomic classification.

Samples were collected from intertidal habitats during low tide. Water,sediment, living plant material and decaying plant/animal debris wereplaced into sterile 50 ml tubes. Portions of each sample along with thewater were spread onto solid agar plates of isolation media. Isolationmedia consisted of: 500 ml of artificial seawater, 500 ml of distilledwater, 1 g of glucose, 1 g of glycerol, 13 g of agar, 1 g of glutamate,0.5 g of yeast extract, 0.5 g casein hydrolysate, 1 ml of a vitaminsolution (100 mg/L thiamine, 0.5 mg/L biotin, 0.5 mg B₁₂), 1 ml of atrace mineral solution (PII metals, containing per liter: 6.0 gFeCl₃6H₂O, 6.84 g H₃BO₃, 0.86 g MnCl₂4H₂O, 0.06 g ZnCl₂, 0.026 CoCl₂6H₂0, 0.052 g NiSO₄H₂O, 0.002 g CuSO₄5H₂O and 0.005 g Na₂MoO₄2H₂O), and 500mg each of penicillin G and streptomycin sulfate. The agar plates wereincubated in the dark at 20-25° C. After 2-4 days the agar plates wereexamined under magnification, and colonies of cells were picked with asterile toothpick and restreaked onto a fresh plate of media. Cells wererepeatedly streaked onto fresh media until contaminated organisms wereremoved.

Colonies from agar plates were transferred to petri dishes withhalf-strength seawater and (1 ml) of a suspension of autoclaved newlyhatched brine shrimp larvae. The brine shrimp larvae became heavilyovergrown with clusters of sporangia after 2-3 days. Released zoosporeswere biflagellate at discharge, swimming actively away from the maturesporangium, wall remnants of which are clearly visible (in phasecontrast) after spore release. Sporangia measured 12.5 μm to 25 μm indiameter, and zoospores were 2.5 μm to 2.8 μm×4.5 μm to 4.8 μm in size.There were 8 to 24 spores per individual sporangium. Settled zoosporesenlarged and rapidly underwent binary divisions leading to tetrads,octads, and finally to clusters of sporangia. Tetrad formation commencedat a very early stage prior to maturity of the sporangia. Thesecharacteristics are in agreement with the genus Schizochytrium.

The isolated thraustochytrid deposited under ATCC Accession No. PTA-9695was further characterized based on the similarity of its 18s rRNA geneto that of known species. Total genomic DNA from the thraustochytriddeposited under ATCC Accession No. PTA-9695 was prepared by standardprocedures (Sambrook J. and Russell D. 2001. Molecular cloning: Alaboratory manual, 3rd edition. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.) and used for PCR amplification of the 18s RNAgene. The PCR amplification of the 18s rRNA gene was carried out withprimers previously described (Honda et. al., J. Eukaryot. Microbiol.46(6) 1999). The PCR conditions with chromosomal DNA template were asfollows: 0.2 μM dNTPs, 0.1 μM each primer, 8% DMSO, 200 ng chromosomalDNA, 2.5 U PfuUltra® II fusion HS DNA polymerase (Stratagene), and 1×PfuUltra® buffer (Stratagene) in a 50 μL total volume. The PCR Protocolincluded the following steps: (1) 95° C. for 2 minutes; (2) 95° C. for45 seconds; (3) 55° C. for 30 seconds; (4) 72° C. for 2 minutes; (5)repeat steps 2-4 for 40 cycles; (6) 72° C. for 5 minutes; and (7) holdat 6° C.

PCR amplification yielded a distinct DNA product with the expected sizeusing chromosomal template described above. The PCR product was clonedinto the vector pJET1.2/blunt (Fermentas) according to themanufacturer's instructions, and the insert sequence was determinedusing supplied standard primers.

Table 2 shows a comparison of the 18s rRNA sequence from thethraustochytrid deposited under ATCC Accession No. PTA-9695 to DNAsequences in the National Center for Biotechnology Information (NCBI)electronic database. Briefly, “% Identity” was determined by the scoringmatrix “swgapdnamt” within the “AlignX” program of the VectorNTI program(Invitrogen), a standard for DNA alignment. The “% Coverage” was takenfrom the results of a Basic Local Alignment Search Tool (BLAST)calculation from the NCBI electronic database and is the percent of thequery length that is included in the aligned segments.

TABLE 2 Comparison of 18s rRNA Sequences % Identity % CoverageThraustochytrids Calculation #1 Calculation #2 Thraustochytriumaggregatum (p) 98 90 Thraustochutriidae sp. HU1 84 86 Thraustochutriidaesp. 8-7 84 91 Thraustochytrium multirudimentale 81 88 Thraustochutriidaesp. PW19 81 85 Schizochytrium sp. ATCC 20888 81 95 (p): indicatespartial sequence

As shown in Table 2, it was found that, in terms of % identity, the 18srRNA gene sequence (SEQ ID NO: 1) from the thraustochytrid depositedunder ATCC Accession No. PTA-9695 is closely related, though notidentical, to the 18s rRNA gene sequence of T. aggregatum provided inHonda, D. et al., J. Euk. Micro. 46(6): 637-647 (1999). The 18s rRNAsequence published for Thraustochytrium aggregatum is a partialsequence, with an approximately 71 DNA nucleotide gap in the middle ofthe sequence. In terms of percent coverage, the 18s rRNA gene sequenceof the isolate of the invention is more closely related toSchizochytrium sp. ATCC 20888 than to T. aggregatum.

Highly conserved proteins such as actin and beta-tubulin have beenwidely used, along with 18s rRNA gene, as markers for assessingphylogenetic relationships between organisms (Baldauf, S. M. Am. Nat.154, S178 (1999)). Total genomic DNA from the thraustochytrid depositedunder ATCC Accession No. PTA-9695 was also used as a template for PCRamplification of both the actin and beta-tubulin genes. The PCRamplification was carried out with primers designed to conserved regionsfrom the actin and beta-tubulin DNA sequences from T. aggregatum.

The PCR conditions with chromosomal DNA template were as follows: 0.2 μMdNTPs, 0.1 uM each primer, 8% DMSO, 200 ng chromosomal DNA, 2.5 UHerculase® II fusion DNA polymerase (Stratagene), and 1× Herculase®buffer (Stratagene) in a 50 μL total volume. The PCR Protocol includedthe following steps: (1) 95° C. for 2 minutes; (2) 95° C. for 30seconds; (3) 55° C. for 30 seconds; (4) 72° C. for 2 minutes; (5) repeatsteps 2-4 for 40 cycles; (6) 72° C. for 5 minutes; and (7) hold at 6° C.

PCR amplification yielded distinct DNA products with the expected sizesusing chromosomal template described above. The respective PCR productswere cloned into the vector pJET1.2/blunt (Fermentas) according to themanufacturer's instructions, and the insert sequence of each weredetermined using supplied standard primers.

Table 3 shows identities for the actin amino acid sequence (SEQ ID NO:3) from the thraustochytrid deposited under ATCC Accession No. PTA-9695as compared to actin sequences available in the public database.Identities were determined through use of the scoring matrix“blosum62mt2” within the “AlignX” program of the VectorNTI program, astandard for protein alignment.

TABLE 3 Comparison of Actin Protein Sequence % IdentitiesThraustochytrids % Identity Thraustochytriidae sp. RT49 98Schizochytrium sp. ATCC 20888 96 Thraustochytrium striatum 96Thraustochytrium aggregatum 96 Japonochytrium marinum 95Thraustochytrium aureum 95

Table 4 shows identities for the beta-tubulin amino acid sequence (SEQID NO: 5) from the thraustochytrid deposited under ATCC Accession No.PTA-9695 as compared to beta-tubulin sequences available in the publicdatabase. Identities were determined through use of the scoring matrix“blosum62mt2” within the “AlignX” program of the VectorNTI program, astandard for protein alignment.

TABLE 4 Comparison of Beta-Tubulin Protein Sequence % IdentitiesThraustochytrids % Identity Aplanochytrium kerguelense 100Aplanochytrium stocchinoi 100 Japonochytrium marinum 100 Labyrinthulasp. N8 100 Thraustochytriidae sp. RT49 100 Thraustochytrium aggregatum100 Thraustochytriidae sp. HU1 100 Thraustochytrium aureum 100Thraustochytrium kinnei 100 Thraustochytriidae sp. #32 100Thraustochytriidae sp. PW19 100 Schizochytrium aggregatum 100Schizochytrium sp. ATCC 20888 100

Based on the above characterizations, the isolated thraustochytriddeposited under ATCC Accession No. PTA-9695 is believed to represent anew Schizochytrium species and is therefore also designated asSchizochytrium sp. ATCC PTA-9695.

EXAMPLE 2

The isolated thraustochytrid deposited under ATCC Accession No. PTA-9695produced high levels of cell growth under varying culture conditions, asdescribed below. Typical media and cultivation conditions are shown inTable 1. Also, high levels of fatty acids and DHA were observed (i.e.,greater than 50% by weight of the dry cell weight were fatty acids andgreater than 50% by weight of the fatty acid methyl esters was DHA).

In carbon and nitrogen-fed cultures with 8200 ppm Cl⁻ at 22.5° C. with20% dissolved oxygen at pH 7.0, the isolate produced a dry cell weightof 140 g/L after 7 days of culture, with a fatty acid content of 70% byweight. Closed loop ammonia feed was used and the pH was maintained at7.0. The omega-3 productivity was 8.92 g/(L*day) under these conditions,with 4.7 g/L EPA (5% by weight of fatty acids) and 56.3 g/L DHA (57% byweight of fatty acids) in 7 days.

In carbon and nitrogen-fed cultures with 3640 ppm Cl⁻ at 22.5° C. with20% dissolved oxygen at pH 7.0, the isolate produced a dry cell weightof 82 g/L after 7 days of culture, with a fatty acid content of 58% byweight. The omega-3 productivity was 4.5 g/(L*day) under theseconditions, with 2.1 g/L EPA (4.3% by weight of fatty acids) and 28.5g/L DHA (58.7% by weight of fatty acids) in 7 days.

In carbon and nitrogen-fed cultures with 980 ppm Cl⁻ at 22.5° C. with20% dissolved oxygen at pH 7.0, the isolate produced a dry cell weightof 60 g/L after 7 days of culture, with a fatty acid content of 53% byweight. The omega-3 productivity was 2.8 g/(L*day) under theseconditions, with 1.1 g/L EPA (3.4% by weight of fatty acids) and 18.4g/L DHA (56.8% by weight of fatty acids) in 7 days.

EXAMPLE 3

Oils were extracted from a biomass sample (Sample A) of the isolatedthraustochytrid deposited under ATCC Accession No. PTA-9695. The biomasssample was produced in a carbon and nitrogen-fed culture with 980 ppmCl⁻ at 22.5° C. with 20% dissolved oxygen at pH 7.0. Oils were extractedfrom biomass Sample A by the hexane extraction process to yieldmicrobial oil Sample A1. Briefly, dried biomass was ground with hexaneusing stainless steel tubes and stainless steel ball bearings forapproximately 2 hours. The slurry was vacuum filtered and the filtratewas collected. The hexane was removed using a rotary evaporator. Oilswere also extracted from biomass Sample A using the FRIOLEX® process(GEA Westfalia Separator UK Ltd., Milton Keynes, England) to yieldmicrobial oil Sample A2. Individual lipid classes were isolated frommicrobial oil Samples A1 and A2 using low pressure flash chromatography,and the weight percent of each class was determined. The fatty acidprofile of each class was determined using gas chromatography with flameionization detection (GC-FID) as fatty acid methyl esters (FAME).

Flash Chromatography—Flash chromatography was used to separate the lipidclasses present in the crude oils, and to determine the weight percentof each class present in the oils. The chromatography system utilizedSilica Gel 60 (EMD Chemical, Gibbstown, N.J.) with mobile phase composedof Petroleum Ether and Ethyl Acetate at 3 mL/min. A step gradient wasused to selectively elute each lipid class from the column. The mobilephase gradient started from 100% petroleum ether and finished with 50%ethyl acetate (followed by a 100% methanol wash). Fractions werecollected in 10 mL test tubes using a Gilson FC 204 large-bed fractioncollector (Gilson, Inc., Middleton, Wis.). Each tube was analyzed bythin layer chromatography (TLC) and the tubes containing individuallipid classes (as judged by single spots on TLC plate with expectedretention factor (Rf)) were pooled, concentrated to dryness, andweighed. The total fraction content was then determined gravimetrically.

TLC Analysis—Thin layer chromatography was conducted on silica gelplates. The plates were eluted using a solvent system consisting ofpetroleum ether: ethyl ether: acetic acid (80:20:1) and were visualizedusing iodine vapor. The Rf values of each spot were then compared withreported literature values for each lipid class.

Fatty Acid Analysis—The samples of biomass and isolated lipid classeswere analyzed for fatty acid composition as FAMEs. Samples were weigheddirectly into screw cap test tubes, and 1 mL of C19:0 internal standard(NuCheck, Elysian, Minn.) in toluene and 2 mL of 1.5 N HCl in methanolwas added to each tube. The tubes were vortexed briefly and placed in aheating block for 2 hours at 100° C. The tubes were removed from theheating block, allowed to cool, and 1 mL of saturated NaCl in water wasadded. The tubes were vortexed again, centrifuged, and a portion of thetop (organic) layer was placed in a GC vial and analyzed by GC-FID.FAME's were quantified using a 3-point internal standard calibrationcurve generated using Nu-Chek-Prep GLC reference standard (Nu-Chek Prep,Inc., Elysian, Minn.) and tentatively identified based on retentiontime. Fatty acids present were expressed as mg/g and % of total FAME.

Sample A1 was prepared by dissolving the crude oil in hexane andapplying to the head of the column. After fractionation of the sampleusing flash chromatography, the sterol ester fraction accounted for 1.2%by weight, the triacylglycerol (TAG) fraction accounted for 82.7% byweight, the free fatty acid (FFA) fraction accounted for 0.9% by weight,and the diacylglycerol (DAG) fraction accounted for 2.9% by weight ofthe crude oil. The total fatty acid profiles of the Sample A1 crude oiland isolated fractions are shown below in Table 5 and Table 6 calculatedas mg/g and % FAME, respectively.

TABLE 5 Sample A1 Fatty Acid Profiles Calculated as Milligrams per GramFAME Biomass Crude Oil Sterol Esters TAG FFA DAG Wt. % NA 38% 1.2% 82.7%0.9 2.9% Fatty Acid FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME(mg/g) FAME (mg/g) C12:0* 0.6 0.0 1.9 3.2 1.7 0.0 C14:0* 5.7 13.6 12.820.2 13.0 17.6 C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 1.3 3.4 3.1 3.1 2.12.6 C16:0* 105.5 239.5 222.2 274.3 183.3 225.1 C16:1* 0.0 0.0 0.8 0.00.8 0.0 C18:0* 6.4 16.4 43.1 16.8 9.8 14.0 C18:1 N9* 0.0 3.8 1.9 3.3 1.03.5 C18:1 N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 0.0 0.0 0.0 0.0 0.0C20:0* 1.8 5.5 13.0 4.7 2.0 2.9 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1N9* 0.0 0.0 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.0 0.6 0.0 C20:2 N6*0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.0 0.87.3 0.8 0.0 1.2 C20:4 N7 0.0 0.0 0.8 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.00.0 0.0 0.0 C20:4N6* 1.0 3.4 0.0 2.6 2.0 1.9 C22:1 N9* 0.0 0.0 0.0 0.00.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 1.5 4.1 1.5 3.5 2.12.1 C20:5 N3* 18.2 39.5 3.5 38.4 30.6 42.8 C24:0* 0.0 0.0 6.3 0.0 0.00.0 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0C22:5 N6* 11.9 29.5 8.9 26.9 14.8 18.7 C22:5 N3* 1.1 4.7 0.9 3.6 3.4 2.7C22:6 N3* 253.5 569.7 107.3 556.5 352.8 451.4 Sum of all FAME's 408.6934.0 435.4 958.0 620.1 786.4

TABLE 6 Sample A1 Fatty Acid Profiles as a Percent of Total FAME Bio-Crude mass Oil Sterol TAG FFA DAG % % Esters % % % Fatty Acid FAME FAME% FAME FAME FAME FAME C12:0* 0.1 0.0 0.4 0.3 0.3 0.0 C14:0* 1.4 1.5 2.92.1 2.1 2.2 C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 0.3 0.4 0.7 0.3 0.3 0.3C16:0* 25.8 25.6 51.0 28.6 29.6 28.6 C16:1* 0.0 0.0 0.2 0.0 0.1 0.0C18:0* 1.6 1.8 9.9 1.8 1.6 1.8 C18:1 N9* 0.0 0.4 0.4 0.3 0.2 0.4 C18:1N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:0* 0.40.6 3.0 0.5 0.3 0.4 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.00.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.0 0.1 0.0 C20:2 N6* 0.0 0.0 0.00.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.0 0.1 1.7 0.1 0.00.1 C20:4 N7 0.0 0.0 0.2 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0C20:4N6* 0.3 0.4 0.0 0.3 0.3 0.2 C22:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 0.4 0.4 0.4 0.4 0.3 0.3 C20:5 N3*4.5 4.2 0.8 4.0 4.9 5.4 C24:0* 0.0 0.0 1.4 0.0 0.0 0.0 C22:4 N9 0.0 0.00.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 2.9 3.2 2.12.8 2.4 2.4 C22:5 N3* 0.3 0.5 0.2 0.4 0.5 0.3 C22:6 N3* 62.0 61.0 24.658.1 56.9 57.4 Sum of 100.0 100.0 100.0 100.0 100.0 100.0 FAME %

Sample A2 was prepared by dissolving the crude oil in hexane andapplying to the head of the column. After fractionation of the sampleusing flash chromatography, the sterol ester fraction accounted for 0.8%by weight, the triacylglycerol (TAG) fraction accounted for 83.4% byweight, the free fatty acid (FFA) fraction accounted for 1.8% by weight,and the diacylglycerol (DAG) fraction accounted for 5.6% by weight ofthe crude oil. The total fatty acid profiles of the Sample A2 crude oiland isolated fractions are shown below in Table 7 and Table 8 calculatedas mg/g and % FAME, respectively.

TABLE 7 Sample A2 Fatty Acid Profiles Calculated as Milligrams per GramFAME Crude Sterol Biomass Oil Esters TAG FFA DAG Wt. % NA NA 0.8% 83.4%1.8% 5.6% FAME FAME FAME FAME FAME FAME Fatty Acid (mg/g) (mg/g) (mg/g)(mg/g) (mg/g) (mg/g) C12:0* 0.6 0.0 0.0 1.5 0.0 1.0 C14:0* 5.7 13.2 8.914.1 9.5 5.4 C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 1.3 3.3 2.8 3.4 2.12.2 C16:0* 105.5 233.7 183.8 246.1 159.7 137.3 C16:1* 0.0 0.0 0.0 0.80.0 0.0 C18:0* 6.4 16.6 23.6 16.9 11.3 5.6 C18:1 N9* 0.0 7.6 5.0 4.3 2.42.6 C18:1 N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 2.2 0.7 1.6 0.8 5.1C20:0* 1.8 5.2 12.1 5.5 2.6 1.1 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1N9* 0.0 0.0 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.8 1.0 0.0 C20:2 N6*0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.3 0.0 0.0 C22:0* 0.0 0.76.0 1.3 0.8 0.0 C20:4 N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.00.0 0.0 0.0 C20:4 N6* 1.0 3.0 0.0 3.1 2.3 1.2 C22:1 N9* 0.0 0.0 0.0 0.00.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 1.5 4.1 1.4 4.3 2.71.0 C20:5 N3* 18.2 38.6 2.7 38.6 39.5 45.5 C24:0* 0.0 0.0 4.7 0.6 0.00.3 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0C22:5 N6* 11.9 28.2 8.6 29.6 18.0 14.7 C22:5 N3* 1.1 3.4 0.0 3.5 2.5 2.2C22:6 N3* 253.5 566.7 102.2 575.0 475.3 447.2 Sum of all 408.6 926.5362.3 951.3 730.4 672.5 FAME's

TABLE 8 Sample A2 Fatty Acid Profiles as a Percent of Total FAME CrudeSterol Oil Esters TAG FFA DAG Biomass % % % % % Fatty Acid % FAME FAMEFAME FAME FAME FAME C12:0* 0.1 0.0 0.0 0.2 0.0 0.2 C14:0* 1.4 1.4 2.41.5 1.3 0.8 C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 0.3 0.4 0.8 0.4 0.3 0.3C16:0* 25.8 25.2 50.7 25.9 21.9 20.4 C16:1* 0.0 0.0 0.0 0.1 0.0 0.0C18:0* 1.6 1.8 6.5 1.8 1.5 0.8 C18:1 N9* 0.0 0.8 1.4 0.5 0.3 0.4 C18:1N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 0.2 0.2 0.2 0.1 0.8 C20:0* 0.40.6 3.3 0.6 0.4 0.2 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.00.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.1 0.1 0.0 C20:2 N6* 0.0 0.0 0.00.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.0 0.1 1.7 0.1 0.10.0 C20:4 N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0C20:4 N6* 0.3 0.3 0.0 0.3 0.3 0.2 C22:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 0.4 0.4 0.4 0.4 0.4 0.2 C20:5N3* 4.5 4.2 0.7 4.1 5.4 6.8 C24:0* 0.0 0.0 1.3 0.1 0.0 0.0 C22:4 N9 0.00.0 0.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 2.9 3.02.4 3.1 2.5 2.2 C22:5 N3* 0.3 0.4 0.0 0.4 0.3 0.3 C22:6 N3* 62.0 61.228.2 60.4 65.1 66.5 Sum of 100.0 100.0 100.0 100.0 100.0 100.0 FAME %

EXAMPLE 4

Triacylglycerides (TAGs) were isolated from a sample of microbial oilpreviously extracted from the isolated thraustochytrid deposited underATCC Accession No. PTA-9695 using hexane extraction (sample A1) or theFRIOLEX® process (GEA Westfalia Separator UK Ltd., Milton Keynes,England) (sample A2), as described in Example 3. The relative areapercent of each TAG isomer was determined using non-aqueousreversed-phase high performance liquid chromatography (NARP-HPLC) withatmospheric pressure chemical ionization-mass spectrometry (APCI-MS)detection, and a tentative identification of each positional isomer wasmade using the mass spectra fragmentation patterns.

Individual lipid classes, including TAGs, were isolated using flashchromatography. The TAG fraction was analyzed by HPLC/APCI-MS todetermine which fatty acid moieties are present in each TAG species, andthe relative amounts of each TAG species. Tentative identification ofeach TAG peak was based on retention time and the APCI spectra of eachpeak. When using NARP-HPLC, the retention of each TAG increases with theequivalent carbon number (ECN), which is defined as the total number ofcarbons in all of the acyl chains minus two times the number of doublebonds. Also, when using optimal chromatographic conditions, criticalpairs of TAG species with the same ECN but with different distributionsof saturated and unsaturated fatty acids, as well as fatty acids withvarying chain lengths can also be resolved. The APCI mass spectra ofeach TAG peak provides the masses of the protonated molecular ion [M+H]⁺, ammonium adduct ions [M+NH₄]⁺ , and DAG fragment ions. Each TAG yieldsa distinctive mass spectrum, and the mass of the DAG fragment ions helpto determine the identity of each TAG species. The fragment ioncorresponding to a loss of an acyl group from the sn-2 position will bethe least intense signal in an APCI spectra because it is energeticallyless favorable than a loss at the sn-1 or sn-3 position. Modern Methodsfor Lipid Analysis by Liquid Chromatography Mass Spectrometry andRelated Techniques 276-297 (William Craig Byrdwell ed., 2005).

Flash Chromatography—Flash chromatography was used to separate the lipidclasses present in the crude oil and to determine the weight percent ofeach class present in the oil. The chromatography system utilized SilicaGel 60 with a mobile phase composed of Petroleum Ether and Ethyl Acetateat 3 mL/min. A step gradient was used to selectively elute each lipidclass from the column. The mobile phase gradient started from 100%petroleum ether and finished with 50% ethyl acetate (followed by a 100%methanol wash). Fractions were collected in 20 mL test tubes using aGilson FC 204 large-bed fraction collector (Gilson, Inc., Middleton,Wis.). Each tube was analyzed by TLC and the tubes containing individuallipid classes (as judged by single spots on TLC plate with expected Rf)were pooled, concentrated to dryness, and weighed. The total fractioncontent was then determined gravimetrically.

TLC Analysis—Thin layer chromatography was conducted on silica gelplates. The plates were eluted using a solvent system consisting ofpetroleum ether: ethyl ether: acetic acid (80:20:1) and were visualizedusing iodine vapor. The Rf values of each spot were then compared withreported literature values for each lipid class.

HPLC/APCI-MS Analysis—The LC/MS system used consisted of a HewlettPackard model 1100 HPLC equipped with atmospheric pressure chemicalionization (APCI) and a Hewlett Packard model 1100 mass selectivedetector (MSD) (Agilent Technologies, Inc., Santa Clara, Calif.). TheHPLC method utilized two PHENOMENEX® C18 column (250 mm×4.6 mm, 5 μm;Phenomenex, Inc. (Torrance, Calif.)) connected in series, a flow rate of1 mL/min., an injection volume of 2 μL, and a column temperature of 50°C. The mobile phase consisted of 0.1% ammonium acetate in isopropanol(Solvent A) and acetonitrile (Solvent B). A linear gradient was usedstarting at 20% Solvent A, increasing to 75% Solvent A in 40 minutes,holding at 75% Solvent A for 5 minutes, returning to 20% Solvent A in 1minute, and holding at 20% Solvent A for an additional 9 minutes. TheMSD mass range was set to m/z 400-1150, with a fragmentor voltage of150, a drying gas flow of 6 L/min, a nebulizer pressure of 45 psig, adrying gas temperature of 350° C., a vaporizer temperature of 325° C., acapillary voltage of 3500 V, and a corona current of 10 μA.

Tridocosahexaenoin (Tri-DHA)—A Tri-DHA STD (NuCheck, Elysian, Minn.) wasused to evaluate the chromatographic system and the accuracy of thedetector response. The retention time of the Tri-DHA peak was 22.5minutes, and the total ion chromatogram (TIC) gave good signal to noiseratio. The APCI mass spectra of the Tri-DHA peak shows the protonatedmolecular ion [M+H]⁺ at m/z 1023.7, the ammonium adduct ions [M+NH₄]⁺ atm/z 1040.8, and a single characteristic DAG fragment ion at m/z 695.5.

A sample of the isolated TAG fraction was prepared in hexane andanalyzed by NARP HPLC/APCI-MS to determine the identities of theindividual TAG isomers.

The mass spectrum of each peak was evaluated and a tentativeidentification of each fatty acid moiety was made, as summarized inTables 9 and 10 below.

TABLE 9 Tentative Identification of TAG Species by LC/APCI-MS in SampleA1 Tentative Retention Identification Area Major (DAG) Time Peak #(sn-1/sn-2/sn-3) Percent [M + H]⁺ [M + NH4]⁺ Fragments 21.4 1EPA/EPA/DHA 0.3 971.7 988.8 643.5, 669.3 21.9 2 DHA/DHA/EPA 4.4 997.81014.7 669.5, 695.5 22.4 3 DHA/DHA/DHA 11.6 1023.7 1040.7 695.5 23.3 4DHA/ARA/DHA 0.5 999.6 1016.7 671.4 23.6 5 DHA/DPA/DHA 0.2 1025.9 1042.8697.5 24.1 6 DHA/DHA/ARA 0.7 999.7 1016.8 671.5, 695.5 24.6 7DHA/DHA/DPA 2.4 1025.7 1042.5 695.5, 697.4 25.3 8 EPA/14:0/DHA 0.3 897.6914.6 569.4, 595.3 25.9 9 DHA/DHA/14:0 2.6 923.7 940.7 595.4, 695.5 27.210 DHA/DHA/15:0 0.4 937.8 954.7 609.5, 695.5 27.5 11 EPA/EPA/16:0 0.2899.5 916.7 597.5, 643.5 28.0 12 DHA/16:0/EPA 4.7 925.8 942.7 597.4,623.5, 669.3 28.5 13 DHA/DHA/16:0 30.8 951.7 968.7 623.7, 695.5 29.5 14DHA/ARA/16:0 0.7 927.6 944.7 599.5, 671.5 29.7 15 DHA/DPA/16:0 0.9 953.7970.8 625.5, 697.5 30.4 16 DHA/16:0/ARA 0.9 927.7 944.6 599.5, 623.530.8 17 DHA/16:0/DPA 4.5 953.8 970.7 623.4, 625.3 31.1 18 DHA/18:0/DHA1.8 979.7 996.7 651.5 32.3 19 DHA/14:0/16:0 2.3 851.7 868.7 523.5 33.620 DHA/15:0/16:0 0.8 865.7 882.7 537.4 DHA/20:0/DHA 1007.8 1024.9 679.534.9 21 DHA/16:0/16:0 19.3 879.7 896.7 551.5, 623.5 36.1* 22DHA/22:0/DHA 0.5 1035.8 1052.8 707.7 DHA/18:0/15:0 893.8 910.8 565.5DPA/16:0/16:0 881.8 898.8 551.5, 625.4 37.4 23 DHA/16:0/18:0 3.7 907.7924.7 579.5, 623.5 38.9 24 16:0/16:0/14:0 0.3 NA 796.7 523.4, 551.5 40.125 DHA Containing 0.4 1117.9 1134.9 789.7 TAG 40.8 26 DHA Containing 0.41091.9 1108.9 763.7 TAG 41.3 27 16:0/16:0/16:0 1.0 NA 824.6 551.5 42.028 DHA/16:0/22:0 0.2 963.8 980.8 623.3, 635.4 43.5* 29 DHA/20:0/22:1 0.31017.8 1034.8 689.7 16:0/20:0/14:0 835.6 852.8 579.5, 607.5 45.6* 30DHA/22:0/22:1 0.7 1045.8 1062.8 717.7 46.3* 31 DHA/20:0/22:0 0.5 1019.81036.9 691.7

TABLE 10 Tentative Identification of TAG Species by LC/APCI-MS in SampleA2 Tentative Identification Retention Time Peak # (sn-1/sn-2/sn-3) AreaPercent [M + H]⁺ [M + NH4]⁺ (DAG) Fragments 21.4 1 EPA/EPA/DHA 0.3 971.7988.8 643.5, 669.3 21.9 2 DHA/DHA/EPA 4.5 997.8 1014.7 669.5, 695.5 22.33 DHA/DHA/DHA 14.0 1023.7 1040.7 695.5 23.3 4 DHA/ARA/DHA 0.6 999.61016.7 671.4 23.6 5 DHA/DPA/DHA 0.3 1025.9 1042.8 697.5 24.1 6DHA/DHA/ARA 0.9 999.7 1016.8 671.5, 695.5 24.6 7 DHA/DHA/DPA 3.0 1025.71042.5 695.5, 697.4 25.4 8 EPA/14:0/DHA 0.2 897.6 914.6 569.4, 595.325.9 9 DHA/DHA/14:0 2.1 923.7 940.7 595.4, 695.5 27.2 10 DHA/DHA/15:00.5 937.8 954.7 609.5, 695.5 27.6 11 EPA/EPA/16:0 0.2 899.5 916.7 597.5,643.5 28.1 12 DHA/16:0/EPA 4.2 925.8 942.7 597.4, 623.5, 669.3 28.5 13DHA/DHA/16:0 31.4 951.7 968.7 623.7, 695.5 29.6 14 DHA/ARA/16:0 0.7927.6 944.7 599.5, 671.5 29.8 15 DHA/DPA/16:0 0.6 953.7 970.8 625.5,697.5 30.4 16 DHA/16:0/ARA 0.9 927.7 944.6 599.5, 623.5 30.8 17DHA/16:0/DPA 4.5 953.8 970.7 623.4, 625.3 31.1 18 DHA/18:0/DHA 2.2 979.7996.7 651.5 32.4 19 DHA/14:0/16:0 1.5 851.7 868.7 523.5 33.6 20DHA/15:0/16:0 0.9 865.7 882.7 537.4 DHA/20:0/DHA 1007.8 1024.9 679.534.9 21 DHA/16:0/16:0 16.1 879.7 896.7 551.5, 623.5 36.1* 22DHA/22:0/DHA 0.4 1035.8 1052.8 707.7 DHA/18:0/15:0 893.8 910.8 565.5DPA/16:0/16:0 881.8 898.8 551.5, 625.4 37.3 23 DHA/16:0/18:0 3.4 907.7924.7 579.5, 623.5 39.7 24 16:0/16:0/14:0 0.9 NA 796.7 523.4, 551.5 40.025 DHA Containing TAG 0.8 1117.9 1134.9 789.7 40.8 26 DHA Containing TAG0.6 1091.9 1108.9 763.7 41.3 27 16:0/16:0/16:0 0.7 NA 824.6 551.5 42.128 DHA/16:0/22:0 0.3 963.8 980.8 623.3, 635.4 43.5* 29 DHA/20:0/22:1 0.21017.8 1034.8 689.7 16:0/20:0/14:0 835.6 852.8 579.5, 607.5 45.6* 30DHA/22:0/22:1 0.8 1045.8 1062.8 717.7 46.3* 31 DHA/20:0/22:0 0.7 1019.81036.9 691.7

EXAMPLE 5

After oil was extracted from the fermentation broth using the Friolexprocess, as described in Example 3, the crude oil was further processedvia refining, bleaching, and deodorizing steps to obtain a final oil.The final oil was diluted with high oleic sunflower oil to obtainfinished commercial oil with a DHA content of approximately 400 mg/g.Individual lipid classes were isolated and the fatty acid profiles ofeach class was determined using gas chromatography with flame ionizationdetection (GC-FID) as fatty acid methyl esters (FAME).

Flash Chromatography—Flash chromatography was used to separate the lipidclasses present in the final oil, and to determine the weight percent ofeach class present in the oil. The chromatography system utilized SilicaGel 60 (EMD Chemical, Gibbstown, N.J.) with mobile phase composed ofPetroleum Ether and Ethyl Acetate at 3 mL/min. A step gradient was usedto selectively elute each lipid class from the column. The mobile phasegradient started from 100% petroleum ether and finished with 50% ethylacetate (followed by a 100% methanol wash). Fractions were collected in10 mL test tubes using a Gilson FC 204 large-bed fraction collector(Gilson, Inc., Middleton, Wis.). Each tube was analyzed by thin layerchromatography (TLC) and the tubes containing individual lipid classes(as judged by single spots on TLC plate with expected retention factor(Rf)) were pooled, concentrated to dryness, and weighed. The totalfraction content was then determined gravimetrically.

TLC Analysis—Thin layer chromatography was conducted on silica gelplates. The plates were eluted using a solvent system consisting ofpetroleum ether: ethyl ether: acetic acid (80:20:1) and were visualizedusing iodine vapor. The Rf values of each spot were then compared withreported literature values for each lipid class.

Fatty Acid Analysis—The final oil sample and isolated lipid classes wereanalyzed for fatty acid composition as FAMEs. Samples were weigheddirectly into screw cap test tubes, and 1 mL of C19:0 internal standard(NuCheck, Elysian, Minn.) in toluene and 2 mL of 1.5 N HCl in methanolwas added to each tube. The tubes were vortexed briefly and placed in aheating block for 2 hours at 100° C. The tubes were removed from theheating block, allowed to cool, and 1 mL of saturated NaCl in water wasadded. The tubes were vortexed again, centrifuged, and a portion of thetop (organic) layer was placed in a GC vial and analyzed by GC-FID.FAME's were quantified using a 3-point internal standard calibrationcurve generated using Nu-Chek-Prep GLC reference standard (Nu-Chek Prep,Inc., Elysian, Minn.) and tentatively identified based on retentiontime. Fatty acids present were expressed as mg/g and % of total FAME.

The sample was prepared by dissolving 250 mg of final oil in 600 μL ofhexane and applying to the head of the column. After fractionation ofthe sample using flash chromatography, the sterol ester fractionaccounted for 1.2% by weight, the triacylglyceride (TAG) fractionaccounted for 92.1% by weight, the free fatty acid (FFA) fractionaccounted for 2.1% by weight, the sterol fraction accounted for 1.1%,the diacylglyceride (DAG) fraction accounted for 2.8% by weight of thefinal oil.

The TLC analysis of the pooled fractions showed that the FFA and sterolfractions were mixed with TAG and DAG respectively. The total fatty acidprofiles of the FRIOLEX® final oil and isolated fractions are shownbelow in Table 11 and Table 12 calculated as mg/g and % FAME,respectively.

TABLE 11 Fatty Acid Profile Calculated as Milligrams per Gram of FAMEFinal Sterol Oil Esters TAG FFA Sterol DAG Wt. % NA 1.2 92.1 2.1 1.1 2.8FAME FAME FAME FAME FAME FAME Fatty Acid (mg/g) (mg/g) (mg/g) (mg/g)(mg/g) (mg/g) C12:0* 0.0 0.0 1.0 0.0 1.2 0.6 C14:0* 11.5 5.1 11.3 6.09.6 5.7 C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 2.3 0.0 2.3 1.2 2.0 1.9C16:0* 183.3 80.0 180.8 99.9 149.3 132.2 C16:1* 0.0 0.0 0.9 0.0 0.8 0.6C18:0* 19.6 17.5 19.6 7.5 16.2 6.7 C18:1 N9* 243.3 242.8 249.6 62.9190.5 84.0 C18:1 N7 1.9 1.7 2.0 0.8 1.9 0.9 C18:2 N6* 13.8 5.6 13.8 6.214.3 9.1 C20:0* 4.3 6.6 4.5 1.5 3.6 1.4 C18:3 N3* 0.0 0.0 0.3 0.0 0.00.0 C20:1 N9* 0.0 0.0 0.8 0.0 0.8 0.0 C18:4 N3 0.0 0.0 0.7 1.3 0.9 0.4C20:2 N6* 0.0 0.0 0.6 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.3 0.0 0.0 0.0C22:0* 3.3 61.0 3.2 1.1 3.0 1.2 C20:4 N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 1.7 0.0 2.3 1.4 1.9 1.3 C22:1 N9*0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 2.44.5 3.0 2.2 2.6 1.3 C20:5 N3* 28.1 3.0 27.7 38.6 25.6 43.2 C24:0* 1.464.3 1.4 0.0 2.0 1.0 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.00.0 0.0 0.0 0.0 C22:5 N6* 20.0 7.6 21.0 10.1 17.2 14.4 C22:5 N3* 2.8 0.03.1 3.7 3.4 2.9 C22:6 N3* 407.1 72.5 417.4 443.6 350.5 428.5 Sum of all936.1 572.1 967.6 688.0 797.3 737.3 FAME's

TABLE 12 Fatty Acid Profiles as a Percent of Total FAME Final Sterol OilEsters TAG FFA Sterol DAG % % % % % % Fatty Acid FAME FAME FAME FAMEFAME FAME C12:0* 0.0 0.0 0.1 0.0 0.2 0.1 C14:0* 1.2 0.9 1.2 0.9 1.2 0.8C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 0.2 0.0 0.2 0.2 0.2 0.3 C16:0* 19.614.0 18.7 14.5 18.7 17.9 C16:1* 0.0 0.0 0.1 0.0 0.1 0.1 C18:0* 2.1 3.12.0 1.1 2.0 0.9 C18:1 N9* 26.0 42.4 25.8 9.1 23.9 11.4 C18:1 N7 0.2 0.30.2 0.1 0.2 0.1 C18:2 N6* 1.5 1.0 1.4 0.9 1.8 1.2 C20:0* 0.5 1.1 0.5 0.20.5 0.2 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.0 0.1 0.0 0.10.0 C18:4 N3 0.0 0.0 0.1 0.2 0.1 0.1 C20:2 N6* 0.0 0.0 0.1 0.0 0.0 0.0C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.4 10.7 0.3 0.2 0.4 0.2 C20:4N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 0.20.0 0.2 0.2 0.2 0.2 C22:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.00.0 0.0 0.0 0.0 C20:4 N3 0.3 0.8 0.3 0.3 0.3 0.2 C20:5 N3* 3.0 0.5 2.95.6 3.2 5.9 C24:0* 0.2 11.2 0.1 0.0 0.2 0.1 C22:4 N9 0.0 0.0 0.0 0.0 0.00.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 2.1 1.3 2.2 1.5 2.2 1.9C22:5 N3* 0.3 0.0 0.3 0.5 0.4 0.4 C22:6 N3* 43.6 12.7 43.1 64.5 44.058.1 Sum of 100 100 100 100 100 100 FAME %

EXAMPLE 6

An analysis of the triacylglycerides (TAGs) of the final oil describedin Example 5 was performed using techniques described in Example 4. Thetentative identification of each fatty acid moiety was made, assummarized in Tables 13 and 14 below.

TABLE 13 Tentative Identification of Major TAG Species Tentative Peakretention Identification Area Time, min. (sn-1/sn-2/sn-3) Percent 22.0DHA/DHA/EPA 3.4 22.5 DHA DHA/DHA 10.4 24.7 DHA/DHA/DPA 2.1 28.2DHA/16:0/EPA 3.4 28.7 DHA/16:0/DHA 23.0 31.0 DHA/16:0/DPA 3.3 35.2DHA/16:0/16:0 11.6 37.6 DHA/16:0/18:0 4.3 40.4 18:1/18:1/18:1 14.0

TABLE 14 Tentative Identification of TAG Species by LC/APCI-MS TentativeIdentification (DAG) Retention Time Peak # (sn-1/sn-2/sn-3) Area Percent[M + H]⁺ [M + NH4]⁺ Fragments 21.5 1 EPA/EPA/DHA 0.4 971.7 988.7 643.5,669.5 22.0 2 DHA/DHA/EPA 3.4 997.8 1014.7 669.4, 695.5 22.5 3DHA/DHA/DHA 10.4 1023.8 1040.7 695.5 23.4 4 DHA/DHA/ARA 0.5 999.8 1016.7671.4, 695.3 23.7 5 DHA/DPA/DHA 0.3 1025.7 1042.7 697.5 24.2 6DHA/DHA/ARA 0.8 999.7 1016.8 671.5, 695.5 24.7 7 DHA/DHA/DPA 2.1 1025.71042.5 695.5, 697.4 25.6 8 EPA/14:0/DHA 0.2 897.7 914.8 569.4, 595.326.1 9 DHA/14:0/DHA 1.4 923.7 940.7 595.5, 695.5 27.4 10 DHA/15:0/DHA0.3 937.8 954.8 609.3 27.7 11 EPA/16:0/EPA 0.2 899.5 916.7 597.5 28.2 12DHA/16:0/EPA 3.4 925.7 942.7 597.5, 623.4, 669.3 28.7 13 DHA/16:0/DHA23.0 951.7 968.7 623.5, 695.5 29.8 14 DHA/16:0/ARA 0.5 927.7 944.8599.5, 623.5 30.0 15 DHA/16:0/DPA 0.7 953.8 970.8 623.4, 625.5 30.6 16DHA/16:0/ARA 0.8 927.7 944.8 599.5, 623.5 31.0 17 DHA/16:0/DPA 3.3 953.7970.7 623.4, 625.5 31.3 18 DHA/18.0/DHA 1.6 979.8 996.8 651.5 32.6 19DHA/14:0/16:0 1.6 851.8 868.8 523.5 33.9 20 DHA/15:0/16:0 0.8 865.7882.7 537.4 DHA/20:0/DHA 1007.8 1024.8 679.5 35.2 21 DHA/16:0/16:0 11.6879.7 896.8 551.5, 623.5 36.4 22 DHA/22:0/DHA 0.5 1035.8 1052.8 707.7DHA/18:0/15:0 893.8 910.8 565.5 DPA/16:0/16:0 881.8 898.8 551.5, 625.437.6 23 DHA/16:0/18:0 4.3 907.8 924.8 579.5, 623.5 40.0 24 DHA/16:0/20:00.8 935.8 952.8 607.6, 623.5 40.4 25 18:1/18:1/18:1 14.0 885.8 902.8603.5 40.7 26 18:1/16:0/18:1 2.2 859.8 876.8 577.5 41.1 27 DHAContaining TAG 1.6 1092.0 1108.8 763.7 42.4 28 24:0 Containing TAG 0.5963.8 980.9 594.5 43.0 29 18:1/18:1/18:0 1.9 887.7 904.8 603.6, 605.645.9 30 DHA/22:0/22:1 0.9 1045.9 1062.9 717.6 46.6 31 DHA/20:0/22:0 0.71019.8 1036.8 691.5

EXAMPLE 7

A two-day old inoculum flask of the isolated thraustochytrid depositedunder ATCC Accession No. PTA-9695 was prepared in a carbon andnitrogen-fed culture with 980 ppm Cl⁻ (thraustochytrid media).

Mutagenesis was carried out according to following procedure:

A sterile T=2 day old flask, approximately 50 ml, was poured into asterile 40 ml glass homogenizer. The culture received 50 plunges in thehomogenizer. The culture was pipeted out and filtered through a sterile50 micron mesh filter, which was placed in a 50 ml sterile tube (themesh was used as a means of retaining the larger clumps of colonieswhile letting the smaller clusters and single cells pass through the 50micron mesh.). The entire concentrated macerate was collected in asterile 50 ml tube. The macerated culture was vortexed and dilutions atlevels up to 1:100 fold were made in tubes containing thraustochytridmedia. The diluted macerate samples were vortexed prior to adding 200 μlof inoculum to a thraustochytrid media agar petri dish, 100×15 mm,containing 4-5 glass beads (3 mm glass beads). Each plate was gentlyagitated in an effort to have the beads spread the inoculum evenlyaround the plate. Beads were dumped off of plates and plates were leftto sit with covers on for approximately 5 minutes to dry. Lights in boththe sterile hood and adjoining areas were turned off as the procedurewas performed in dim light. There was minimal light available to be ableto run the procedure but only indirect and dim.

Five replicate plates were placed on the floor of the XL crosslinker(Spectronics Corporation, New York) with the lids off while the sampleswere irradiated. The crosslinker delivered power in terms of microjoulesand a level was sought that achieved a 90%-95% Kill. Five replicatecontrol plates were inoculated with un-mutagenized cells using the sameprotocol. These cell counts were used to calculate the % Kill. Once theirradiation was finished the plates were taken out, the lids werereplaced, and the plates were wrapped in parafilm followed by a wrap inaluminum foil. It was imperative that the plates grew for the first weekin the dark so that they were not able to repair the damaged genes.

Plates were placed in a 22.5° C. room for about 10 days prior tocounting the colonies. When final counts were made, individual colonieswere picked with a sterile inoculating loop and re-streaked on newthraustochytrid media plates. Each colony was plated on an individualplate. As plates grew dense a sample was taken, using a inoculatingloop, and inoculated into a sterile 250 ml shake flask containing 50 mlof thraustochytrid media. This flask was placed on a shaker at 200 rpmin a 22.5° C. room. On T=7 days the shake flask culture was harvestedinto a 50 ml sterile tube. The pH was taken and the sample was spun downto collect the biomass pellet. Each sample was rinsed and re-suspendedin a 50:50 mixture of isopropyl alcohol and distilled water prior tobeing re-spun. The collected pellet was freeze dried, weighed, and aFAME analysis was performed. The data in Tables 15-21 represents mutantsproduced with the above process.

TABLE 15 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control Fatty Acids ATCC PTA-9695 Mutant 1 Mutant 2 Mutant 3 Mutant 4Mutant 5 Mutant 8 Mutant 9 Mutant 10 % 08:0 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00% 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 12:0 0.10 0.10 0.08 0.08 0.13 0.07 0.11 0.08 0.08 %12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.11 0.170.13 0.12 0.18 0.11 0.15 0.14 % 13:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 14:0 1.79 1.85 1.49 1.37 2.36 1.29 1.85 1.72 1.57 % 14:10.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 16:0 30.98 28.75 29.96 29.97 30.33 29.8630.97 30.11 29.20 % 16:1 0.27 0.20 0.31 0.14 0.25 0.27 0.16 0.27 0.24 %16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.15 0.13 0.17 0.27 0.12 0.160.13 0.13 % 18:0 1.29 1.22 1.38 1.47 1.22 1.57 1.25 1.34 1.34 % 18:1 n-90.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:1 n-7 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 18:3 n-6 0.00 0.03 0.00 0.00 0.07 0.00 0.03 0.00 0.00 % 18:3n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:0 0.39 0.36 0.42 0.45 0.34 0.460.37 0.40 0.40 % 20:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.38 0.32 0.42 0.44 0.32 0.41 0.330.36 % 20:4 ARA 0.55 0.55 0.94 0.57 0.80 0.89 0.60 0.73 0.75 % 20:5 n-3EPA 2.62 2.94 3.01 2.40 3.64 2.83 2.54 2.81 2.81 % 22:0 0.08 0.08 0.090.09 0.07 0.10 0.07 0.09 0.09 % 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:30.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 22:5 n-6 3.19 3.19 2.94 3.43 3.35 2.873.34 3.01 3.15 % 22:5 n-3 0.18 0.18 0.21 0.23 0.20 0.18 0.20 0.17 0.18 %22:6 n-3 DHA 56.88 58.63 57.56 57.85 54.87 57.98 56.62 57.53 58.52 %24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.08 0.000.00 0.00 0.00 0.00 0.09 0.00 % Fat 46.83 46.10 31.23 47.39 49.78 30.6254.71 37.72 37.87 % Unknown 0.85 0.46 0.35 0.51 0.51 0.36 0.50 0.38 0.39

TABLE 16 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control ATCC Fatty Acids PTA-9695 Mutant 11 Mutant 13 Mutant 14 Mutant15 Mutant 16 Mutant 20 Mutant 21 Mutant 22 % 08:0 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 % 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 12:0 0.10 0.10 0.08 0.09 0.11 0.11 0.09 0.09 0.10% 12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.150.16 0.14 0.13 0.12 0.17 0.16 0.13 % 13:1 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 14:0 1.79 1.89 1.43 1.75 1.83 1.98 1.76 1.77 1.81 %14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 16:0 30.98 31.08 30.27 29.92 31.79 30.1828.84 30.05 30.81 % 16:1 0.27 0.32 0.26 0.28 0.21 0.24 0.23 0.23 0.33 %16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.24 0.15 0.13 0.15 0.12 0.140.16 0.14 % 18:0 1.29 1.36 1.44 1.31 1.36 1.21 1.28 1.34 1.33 % 18:1 n-90.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:1 n-7 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 18:3 n-6 0.00 0.05 0.00 0.00 0.00 0.03 0.00 0.00 0.00 % 18:3n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:0 0.39 0.38 0.42 0.39 0.40 0.370.37 0.38 0.38 % 20:1 n-9 0.00 0.00 0.06 0.00 0.00 0.00 0.00 0.00 0.00 %20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.43 0.36 0.33 0.36 0.37 0.33 0.350.34 % 20:4 ARA 0.55 0.79 0.72 0.80 0.64 0.62 0.83 0.73 0.69 % 20:5 n-3EPA 2.62 3.17 2.72 2.97 2.52 2.66 3.03 2.90 2.87 % 22:0 0.08 0.08 0.090.08 0.08 0.08 0.08 0.08 0.08 % 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:30.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 22:5 n-6 3.19 3.25 3.06 2.97 3.07 3.162.98 3.01 3.02 % 22:5 n-3 0.18 0.20 0.19 0.17 0.19 0.16 0.17 0.18 0.18 %22:6 n-3 DHA 56.88 55.17 57.52 57.63 56.02 57.38 58.58 57.45 56.65 %24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.000.00 0.00 0.07 0.00 0.00 0.08 % Fat 46.83 46.19 37.00 38.41 48.46 47.3237.71 40.23 43.55 % Unknown 0.85 0.47 0.39 0.36 0.47 0.44 0.37 0.39 0.38

TABLE 17 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control ATCC Fatty Acids PTA-9695 Mutant 24 Mutant 26 Mutant 27 Mutant29 Mutant 30 Mutant 33 Mutant 34 Mutant 35 % 08:0 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 % 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 12:0 0.10 0.11 0.09 0.09 0.08 0.08 0.10 0.11 0.09% 12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.120.13 0.14 0.16 0.14 0.12 0.12 0.10 % 13:1 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 14:0 1.79 1.98 1.71 1.69 1.63 1.66 1.93 2.01 1.59 %14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.00 0.000.00 0.00 0.00 0.70 0.54 0.39 % 16:0 30.98 30.61 30.32 30.21 29.70 29.5030.26 32.28 30.78 % 16:1 0.27 0.19 0.22 0.22 0.26 0.26 0.29 0.26 0.16 %16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.15 0.18 0.16 0.13 0.13 0.260.16 0.12 % 18:0 1.29 1.24 1.31 1.31 1.32 1.30 1.32 1.37 1.34 % 18:1 n-90.00 0.00 0.00 0.00 0.00 0.00 0.10 0.11 0.09 % 18:1 n-7 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 18:3 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:0 0.39 0.37 0.39 0.40 0.40 0.390.37 0.40 0.40 % 20:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.13 0.14 %20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.38 0.37 0.35 0.35 0.35 0.00 0.000.00 % 20:4 ARA 0.55 0.61 0.59 0.69 0.68 0.32 0.34 0.24 0.28 % 20:5 n-3EPA 2.62 2.62 2.70 2.85 2.90 2.91 3.28 2.51 2.59 % 22:0 0.08 0.08 0.080.08 0.09 0.08 0.08 0.08 0.08 % 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:30.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 22:5 n-6 3.19 3.10 3.11 3.05 3.10 3.113.43 3.26 3.56 % 22:5 n-3 0.18 0.16 0.18 0.19 0.18 0.18 0.18 0.15 0.24 %22:6 n-3 DHA 56.88 57.03 57.46 57.46 57.96 58.52 55.92 54.96 56.73 %24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.08 0.000.00 0.00 0.00 0.07 0.07 0.07 % Fat 46.83 47.80 43.50 38.86 38.60 38.1646.95 46.43 51.55 % Unknown 0.85 0.45 0.42 0.39 0.37 0.82 1.25 1.23 1.25

TABLE 18 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control ATCC Fatty Acids PTA-9695 Mutant 36 Mutant 37 Mutant 38 Mutant39 Mutant 40 Mutant 42 Mutant 43 Mutant 44 % 08:0 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 % 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 12:0 0.10 0.00 0.11 0.00 0.11 0.09 0.08 0.12 0.09% 12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.440.09 0.24 0.12 0.11 0.12 0.08 0.15 % 13:1 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 14:0 1.79 1.25 1.99 1.48 1.96 1.76 1.43 2.17 1.75 %14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 2.12 0.480.71 0.54 0.55 0.36 0.62 0.50 % 16:0 30.98 26.95 28.04 32.28 30.84 30.2525.77 43.37 30.18 % 16:1 0.27 0.00 0.26 0.23 0.22 0.21 0.10 1.05 0.22 %16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.95 0.13 0.28 0.16 0.16 0.100.26 0.13 % 18:0 1.29 1.58 1.11 1.79 1.30 1.29 1.25 2.21 1.34 % 18:1 n-90.00 0.37 0.08 0.25 0.09 0.09 0.12 0.09 0.10 % 18:1 n-7 0.00 0.00 0.000.00 0.00 0.00 0.00 0.05 0.00 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 18:3 n-6 0.00 0.00 0.06 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:0 0.39 0.34 0.31 0.43 0.38 0.390.36 0.61 0.40 % 20:1 n-9 0.00 0.00 0.00 0.43 0.00 0.14 0.15 0.15 0.49 %20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 % 20:4 ARA 0.55 0.41 0.31 0.24 0.27 0.24 0.30 0.35 0.23 % 20:5 n-3EPA 2.62 5.36 2.77 4.00 2.72 2.80 3.21 3.47 2.80 % 22:0 0.08 0.00 0.070.14 0.07 0.08 0.07 0.14 0.08 % 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:30.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.00 0.060.00 0.00 0.00 0.00 0.00 0.00 % 22:5 n-6 3.19 2.40 3.94 2.57 3.48 3.293.89 2.37 3.33 % 22:5 n-3 0.18 0.00 0.19 0.00 0.17 0.17 0.30 0.33 0.17 %22:6 n-3 DHA 56.88 57.52 58.57 54.20 56.24 57.09 60.99 41.61 56.76 %24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.080.00 0.08 0.09 0.08 0.06 0.09 % Fat 46.83 12.73 54.86 18.08 45.74 42.5942.48 56.44 41.20 % Unknown 0.85 0.29 1.36 0.73 1.28 1.20 1.31 0.90 1.20

TABLE 19 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control ATCC PTA- Fatty Acids 9695 Mutant 45 Mutant 46 Mutant 47 Mutant48 Mutant 49 Mutant 50 Mutant 51 Mutant 52 % 08:0 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 % 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 % 12:0 0.10 0.10 0.13 0.11 0.07 0.09 0.09 0.09 0.11% 12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.110.10 0.09 0.13 0.09 0.13 0.10 0.09 % 13:1 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 14:0 1.79 1.79 2.07 1.86 1.52 1.62 1.78 1.78 1.85 %14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.41 0.760.57 0.46 0.48 0.55 0.53 0.53 % 16:0 30.98 28.79 24.90 30.07 29.07 31.2130.46 30.79 32.53 % 16:1 0.27 0.19 0.24 0.18 0.17 0.17 0.18 0.21 0.22 %16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.11 0.24 0.16 0.12 0.14 0.170.18 0.15 % 18:0 1.29 1.24 1.07 1.28 1.41 1.43 1.36 1.48 1.35 % 18:1 n-90.00 0.08 0.07 0.09 0.09 0.08 0.10 0.09 0.06 % 18:1 n-7 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 18:3 n-6 0.00 0.00 0.12 0.05 0.00 0.00 0.00 0.00 0.00 % 18:3n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:0 0.39 0.36 0.29 0.37 0.42 0.420.39 0.40 0.41 % 20:1 n-9 0.00 0.15 0.13 0.11 0.24 0.13 0.19 0.16 0.19 %20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.05 0.00 0.000.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.00 0.12 0.00 0.00 0.00 0.00 0.000.00 % 20:4 ARA 0.55 0.29 0.65 0.26 0.18 0.21 0.22 0.24 0.24 % 20: n-32.62 3.05 4.28 2.66 2.93 2.46 2.71 2.94 2.44 EPA % 22:0 0.08 0.07 0.060.07 0.09 0.09 0.08 0.08 0.08 % 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:30.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.06 0.070.05 0.00 0.00 0.00 0.00 0.00 % 22:5 n-6 3.19 3.59 4.28 3.46 3.07 3.323.17 3.18 3.24 % 22:5 n-3 0.18 0.25 0.27 0.18 0.17 0.17 0.16 0.17 0.17 %22:6 n-3 DHA 56.88 57.74 58.32 56.70 58.65 56.45 56.83 56.19 55.06 %24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.07 0.150.10 0.10 0.11 0.10 0.10 0.07 % Fat 46.83 48.91 58.95 54.80 35.41 48.6044.93 43.01 51.93 % Unknown 0.85 1.55 1.63 1.57 1.09 1.35 1.31 1.28 1.19

TABLE 20 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control ATCC Fatty Acids PTA-9695 Mutant 53 Mutant 54 Mutant 55 Mutant56 Mutant 57 Mutant 58 Mutant 60 Mutant 61 Mutant 65 % 08:0 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 % 11:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %11:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 12:0 0.10 0.090.08 0.12 0.08 0.08 0.08 0.08 0.10 0.08 % 12:1 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.11 0.12 0.08 0.09 0.13 0.16 0.140.09 0.14 % 13:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %14:0 1.79 1.74 1.63 2.13 1.67 1.59 1.59 1.59 1.85 1.58 % 14:1 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.53 0.52 0.48 0.510.52 0.45 0.50 0.51 0.48 % 16:0 30.98 30.13 29.54 33.01 31.08 29.3730.65 29.39 31.15 30.03 % 16:1 0.27 0.21 0.23 0.26 0.26 0.14 0.25 0.220.26 0.25 % 16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %16:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.150.14 0.14 0.14 0.16 0.12 0.13 0.14 0.13 % 18:0 1.29 1.30 1.30 1.37 1.381.37 1.46 1.30 1.30 1.35 % 18:1 n-9 0.00 0.08 0.08 0.00 0.06 0.11 0.090.10 0.07 0.07 % 18:1 n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-60.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 20:0 0.39 0.38 0.39 0.40 0.42 0.38 0.430.39 0.39 0.41 % 20:1 n-9 0.00 0.19 0.16 0.13 0.19 0.20 0.17 0.14 0.130.21 % 20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-90.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 20:4 ARA 0.55 0.25 0.21 0.26 0.22 0.250.51 0.20 0.24 0.19 % 20:5 n-3 EPA 2.62 2.75 2.78 2.81 2.67 2.78 5.762.72 2.59 2.82 % 22:0 0.08 0.08 0.08 0.08 0.09 0.08 0.09 0.08 0.08 0.09% 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:2 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.06 0.00 % 22:5 n-6 3.19 3.47 3.20 3.25 3.19 3.43 2.62 3.303.42 3.18 % 22:5 n-3 0.18 0.18 0.18 0.17 0.17 0.20 0.59 0.17 0.17 0.17 %22:6 n-3 DHA 56.88 56.99 58.07 54.04 56.38 57.76 54.09 58.21 55.91 57.56% 24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.000.09 0.09 0.07 0.10 0.09 0.11 0.10 0.07 0.08 % Fat 46.83 45.83 39.5948.81 41.92 43.97 33.96 36.97 50.40 36.21 % Unknown 0.85 1.28 1.19 1.191.29 1.35 0.77 1.24 1.48 1.17

TABLE 21 Mutants of Thraustochytrid Strain ATCC Accession No. PTA-9695control Mutant 68 Mutant 70 Mutant 72 Fatty ATCC PTA- Mutant ATCC PTA-ATCC PTA- ATCC PTA- Acids 9695 66 Mutant 67 9696 Mutant 69 9697 Mutant71 9698 Mutant 73 Mutant 74 % 08:0 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00% 10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 12:0 0.15 0.00 0.00 0.00 0.00 0.00 0.000.13 0.00 0.00 % 12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00% 13:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:1 0.000.00 0.00 0.00 0.00 0.00 0.22 0.00 0.00 0.00 % 14:0 2.42 2.29 2.07 2.092.11 2.21 2.27 2.29 1.97 2.05 % 14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.190.00 0.00 0.00 % 15:1 0.55 0.47 0.48 0.47 0.47 0.44 0.46 0.40 0.50 0.47% 16:0 39.19 31.02 26.20 25.84 27.79 28.14 28.89 33.49 24.50 23.95 %16:1 0.43 0.19 0.00 0.00 0.00 0.00 0.19 0.21 0.00 0.00 % 16:2 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 % 17:0 0.16 0.00 0.00 0.00 0.00 0.00 0.00 0.130.00 0.00 % 18:0 1.67 1.68 1.22 1.22 1.44 1.49 1.51 2.24 1.11 1.02 %18:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9 % 18:1 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-7 % 18:2 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 n-6 % 18:3 0.00 0.18 0.20 0.21 0.19 0.17 0.22 0.160.22 0.22 n-3 % 18:4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00n-3 % 20:0 0.49 0.41 0.32 0.31 0.35 0.37 0.44 0.52 0.29 0.27 % 20:1 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9 % 20:2 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 n-9 % 20:3 0.00 0.00 0.00 0.00 0.15 0.00 0.00 0.000.00 0.00 n-6 % 20:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00n-3 % 20:4 0.18 0.16 0.33 0.27 0.24 0.37 0.30 0.27 0.38 0.39 ARA % 20:51.76 2.30 3.86 3.97 3.32 4.12 3.09 2.74 4.43 4.53 n-3 EPA % 22:0 0.330.46 0.35 0.44 0.48 0.38 0.43 0.12 0.35 0.34 % 22:1 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00% 22:4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6 % 22:5 2.622.83 3.17 2.66 2.72 2.95 3.46 2.79 3.17 3.19 n-6 % 22:5 0.18 0.18 0.460.42 0.34 0.61 0.25 0.27 0.48 0.57 n-3 % 22:6 49.52 57.01 60.60 61.4259.74 58.03 55.62 53.06 61.83 62.23 n-3 DHA % 24:0 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 % Fat 52.70 49.32 48.51 49.49 48.80 53.65 40.38 63.4048.27 46.63 % 0.35 0.82 0.73 0.66 0.67 0.73 2.46 1.18 0.78 0.76 Unknown

EXAMPLE 8

Four thraustochytrid samples were obtained from the American Tissue andCulture Collection (ATCC) and each sample was analyzed for the fattyacid profile of the biomass, the fatty acid profile of the extractedcrude oil, the triacylglyceride (TAG) fraction of the crude oil, and thepolar lipid (PL) fraction of the crude oil. The samples analyzed wereATCC 34304, 20890, 20889, and 20892. The strains were inoculated into250 ml shake flask containing 50 mls of the following medium: 1 gpeptone, 1 g yeast extract, 5 g glucose in 1 liter of artificialseawater. Cultures were incubated at 20° C. with shaking at 200 rpm onan orbital shaker. After 7 days the cultures was harvested bycentrifugation (5087×g), washed with a mixture of water: isopropanol(1:1), and centrifuged again. The resulting pellet was freeze dried. Thecrude oil was extracted from the dried biomass using the method of Blighand Dyer (Can. J of Biol. And Phys. 37: 911-917 (1959)). TAGs and PLswere isolated from the crude oil using a variation of the solid phaseextracted (SPE) method developed by Kaluzny et al. (J. Lipid Res. 26:135-140 (1959)). The crude oil and isolated fractions were analyzed forDHA and EPA content as well as the total fatty acid content (as fattyacid methyl esters).

Lipid Extraction—Crude oil was extracted from the freeze dried biomassby weighing 100 to 200 mg into a 1.5×10 cm screw top test tube, adding 8mL of a monophasic system consisting of 1:2:0.8chloroform:methanol:water (CHCl₃:MeOH:H₂O), and homogenizing with aPOLYTRON® PT 3100 dispersing unit equipped with a PT-DA 3012/2aggregate. The sample was homogenized for 2 minutes at 10000 rpm whileimmersed in an ice bath. A biphasic system was produced by adding 2.1 mLof CHCl₃, vortexing for 1 minute, adding 1.7 mL of H₂O, and vortexingagain for an additional 1 minute. The bottom (organic) layer was removedusing a Pasteur pipet and placed into a collection flask. The MeOH-H₂Olayer left in the test tube was re-extracted two more times with 2.1 mLportions of CHCl₃. The organic layers were combined and dried under astream of nitrogen.

Solid Phase Extraction—The TAG and PL fractions were separated from thecrude lipid by SPE using 500 mg aminopropyl cartridges (Burdick &Jackson) placed in a Vac Elut apparatus. The cartridge was conditionwith 5 mL of hexane, and 10 to 20 mg of each sample was dissolved in 400μL CHCl₃ and applied to the cartridge. The column was washed with 4 mLof 2:1 CHCl₃:isopropyl alcohol (IPA) to elute all the neutral lipids,which were collected and dried under nitrogen. The fatty acids were theneluted with 5 mL of 2% acetic acid (HOAc) in ether, which was discarded.The PL portion was eluted with 5 mL of MeOH, which was collected anddried under nitrogen. The neutral lipid fraction was re-dissolved in 400μL of hexane and applied to a second aminopropyl column (previouslyconditioned with 5 mL of hexane). Sterol esters were eluted with 5 mL of1% ethyl acetate (EtOAc) in hexane and discarded. Finally, TAGs wereeluted with 5 mL of 3% EtOAc in hexane, which was collected and driedunder nitrogen.

TLC Analysis—Thin layer chromatography was conducted on silica gelplates. The plates were eluted using a solvent system consisting ofpetroleum ether: ethyl ether: acetic acid (80:20:1) and were visualizedusing iodine vapor.

Fatty Acid Analysis—Samples of biomass, crude oil, isolated TAG, and PLfractions were analyzed for fatty acid composition as FAMEs. Sampleswere weighed directly into screw cap test tubes, and 1 mL of C19:0internal standard in toluene and 2 mL of 1.5 N HCl in methanol was addedto each tube. The tubes were vortexed briefly and placed in a heatingblock for 2 hours at 100° C. The tubes were removed from the heatingblock, allowed to cool, and 1 mL of saturated NaCl in water was added.The tubes were vortexed again, centrifuged, and a portion of the top(organic) layer was placed in a GC vial and analyzed by GC-FID. FAME'swere quantified using a 3-point internal standard calibration curvegenerated using Nu-Chek-Prep GLC reference standard and tentativelyidentified based on retention time. Fatty acids present were expressedas mg/g and % of total FAME.

ATCC 34304—The lipid content of the ATCC 34304 biomass was estimated tobe 9.1% as the sum of FAME, and the amount of crude oil obtained aftersolvent extraction was 9.2% by weight, giving a 101% recovery of fatpresent in the biomass. The EPA and DHA content of the biomass wasdetermined to be 4.8 mg/g and 38.7 mg/g, respectively. The extractedcrude oil contained 25.9 mg/g EPA and 238.7 mg/g DHA. The isolated TAGcontained 13.9 mg/g EPA and 303.9 mg/g DHA, while the isolated PLcontained 38.7 mg/g EPA and 237.980 mg/g DHA. The total fatty acidprofiles of the biomass, extracted crude oil, TAG fraction, and PLfraction are shown below in Table 22 and Table 23 calculated as mg/g and% FAME, respectively.

TABLE 22 Fatty Acid Profile of ATCC 34304 Calculated as Milligrams perGram Biomass Crude Oil TAG PL FAME FAME FAME FAME Fatty Acid (mg/g)(mg/g) (mg/g) (mg/g) C12:0* 0.0 0.0 0.0 0.0 C14:0* 1.5 8.5 13.7 1.5C14:1* 0.0 0.2 0.0 0.0 C15:0 0.0 0.0 0.0 0.0 C16:0* 20.8 138.9 250.873.9 C16:1* 0.2 2.9 6.9 0.3 C18:0* 2.1 17.8 45.6 0.8 C18:1 N9* 3.8 29.874.2 4.2 C18:1 N7 0.0 0.0 0.0 0.0 C18:2 N6* 1.5 12.0 31.0 2.5 C20:0* 0.10.7 1.7 0.1 C18:3 N3* 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.1 0.3 0.0 C18:4 N30.0 8.5 0.3 0.0 C20:2 N6* 0.1 0.8 2.2 0.0 C20:3 N6 0.0 2.5 6.3 0.5C22:0* 0.3 5.2 0.3 0.0 C20:4 N7 0.4 2.7 0.0 0.0 C20:3 N3 0.0 0.0 0.6 0.0C20:4N6* 3.4 20.8 20.9 27.5 C22:1 N9* 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.00.0 0.0 C20:4 N3 0.1 0.6 1.3 0.3 C20:5 N3* 4.8 25.9 13.9 38.7 C24:0* 0.40.2 0.3 0.0 C22:4 N9 0.0 1.2 0.7 0.4 C24:1 N9* 0.9 6.9 18.2 0.8 C22:5N6* 11.0 60.8 51.9 45.9 C22:5 N3* 0.4 3.1 6.8 1.0 C22:6 N3* 38.7 238.7303.9 237.9 Sum of all 91.2 590.9 855.7 437.3 FAME

TABLE 23 Fatty Acid Profiles of ATCC 34304 Calculated as a Percent ofTotal FAME Biomass Crude Oil TAG PL Fatty Acid % FAME % FAME % FAME %FAME C12:0* 0.0 0.0 0.0 0.0 C14:0* 1.7 1.5 1.6 0.4 C14:1* 0.0 0.0 0.00.0 C15:0 0.0 0.0 0.0 0.0 C16:0* 22.8 24.2 29.3 16.9 C16:1* 0.2 0.5 0.80.1 C18:0* 2.3 3.1 5.3 0.2 C18:1 N9* 4.2 5.2 8.7 1.0 C18:1 N7 0.0 0.00.0 0.0 C18:2 N6* 1.6 2.1 3.6 0.6 C20:0* 0.1 0.1 0.2 0.0 C18:3 N3* 0.00.0 0.0 0.0 C20:1 N9* 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.0 C20:2 N6*0.1 0.1 0.3 0.0 C20:3 N6 0.0 0.4 0.7 0.1 C22:0* 0.3 0.9 0.0 0.0 C20:4 N70.5 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.1 0.0 C20:4N6* 3.7 3.4 2.4 6.3 C22:1N9* 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 C20:4 N3 0.1 0.1 0.2 0.1C20:5 N3* 5.3 4.5 1.6 8.8 C24:0* 0.5 0.0 0.0 0.0 C22:4 N9 0.0 0.0 0.10.1 C24:1 N9* 1.0 1.2 2.1 0.2 C22:5 N6* 12.1 10.5 6.1 10.5 C22:5 N3* 0.50.5 0.8 0.2 C22:6 N3* 42.5 41.5 35.5 54.4 Sum of FAME % 100.0 100.0100.0 100.0

ATCC 20890—The lipid content of the ATCC 20890 biomass was estimated tobe 9.2% as the sum of FAME, and the amount of crude oil obtained aftersolvent extraction was 10.2% by weight, giving a 111% recovery of fatpresent in the biomass. The EPA and DHA content of the biomass wasdetermined to be 12.2 mg/g and 36.6 mg/g, respectively. The extractedcrude oil contained 64.7 mg/g EPA and 194.2 mg/g DHA. The isolated TAGcontained 41.9 mg/g EPA and 230.2 mg/g DHA, while the isolated PLcontained 54.4 mg/g EPA and 149.5 mg/g DHA. The total fatty acidprofiles of the biomass, extracted crude oil, TAG fraction, and PLfraction are shown below in Table 24 and Table 25 calculated as mg/g and% FAME, respectively.

TABLE 24 Fatty Acid Profile of ATCC 20890 Calculated as Milligrams perGram Biomass Crude Oil TAG PL FAME FAME FAME FAME Fatty Acid (mg/g)(mg/g) (mg/g) (mg/g) C12:0* 0.0 0.0 0.0 0.0 C14:0* 0.6 5.2 22.6 3.2C14:1* 0.0 0.0 0.0 0.2 C15:0 0.0 0.0 0.0 0.0 C16:0* 25.4 161.1 206.1142.5 C16:1* 0.2 1.3 4.7 0.8 C18:0* 1.8 12.1 58.9 7.1 C18:1 N9* 1.0 9.737.6 2.4 C18:1 N7 0.0 0.0 0.0 0.0 C18:2 N6* 0.8 4.8 6.9 3.9 C20:0* 0.10.0 0.0 0.3 C18:3 N3* 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.0 0.0 0.0 C18:4 N30.0 0.0 0.0 0.0 C20:2 N6* 1.9 11.1 14.2 13.1 C20:3 N6 1.0 5.4 2.4 8.0C22:0* 0.0 0.0 0.0 0.0 C20:4 N7 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0C20:4N6* 5.3 33.6 35.8 35.3 C22:1 N9* 0.0 0.0 0.0 0.0 C20:4 N5 0.2 0.00.0 1.1 C20:4 N3 0.1 2.2 0.0 3.1 C20:5 N3* 12.2 64.7 41.9 54.4 C24:0*0.3 0.0 0.0 0.2 C22:4 N9 0.0 0.0 0.0 0.0 C24:1 N9* 0.3 1.4 1.6 1.9 C22:5N6* 0.5 2.4 1.4 3.0 C22:5 N3* 3.6 19.4 23.9 19.8 C22:6 N3* 36.6 194.2230.2 149.5 Sum of all 92.3 535.1 735.0 450.9 FAME

TABLE 25 Fatty Acid Profiles of ATCC 20890 Calculated as a Percent ofTotal FAME Biomass Crude Oil TAG PL Fatty Acid % FAME % FAME % FAME %FAME C12:0* 0.0 0.0 0.0 0.0 C14:0* 0.7 1.0 3.1 0.7 C14:1* 0.0 0.0 0.00.0 C15:0 0.0 0.0 0.0 0.0 C16:0* 27.6 30.1 28.0 31.6 C16:1* 0.2 0.3 0.60.2 C18:0* 2.0 2.3 8.0 1.6 C18:1 N9* 1.1 1.8 5.1 0.5 C18:1 N7 0.0 0.00.0 0.0 C18:2 N6* 0.9 0.9 0.9 0.9 C20:0* 0.1 0.0 0.0 0.1 C18:3 N3* 0.00.0 0.0 0.0 C20:1 N9* 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.0 C20:2 N6*2.1 2.1 1.9 2.9 C20:3 N6 1.1 1.0 0.3 1.8 C22:0* 0.0 0.0 0.0 0.0 C20:4 N70.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 C20:4N6* 5.8 6.3 4.9 7.8 C22:1N9* 0.0 0.0 0.0 0.0 C20:4 N5 0.2 0.0 0.0 0.3 C20:4 N3 0.1 0.4 0.0 0.7C20:5 N3* 13.2 12.1 5.7 12.1 C24:0* 0.3 0.0 0.0 0.0 C22:4 N9 0.0 0.0 0.00.0 C24:1 N9* 0.3 0.3 0.2 0.4 C22:5 N6* 0.6 0.4 0.2 0.7 C22:5 N3* 3.93.6 3.2 4.4 C22:6 N3* 39.7 36.3 31.3 33.2 Sum of FAME % 100.00 100.0100.0 100.0

ATCC 20889—The lipid content of the biomass was estimated to be 3.3% asthe sum of FAME, and the amount of crude oil obtained after solventextraction was 3.4% by weight, giving a 103% recovery of fat present inthe biomass. The EPA and DHA content of the biomass was determined to be2.3 mg/g and 16.5 mg/g, respectively. The extracted crude oil contained26.8 mg/g EPA and 205.1 mg/g DHA. The isolated TAG contained 7.3 mg/gEPA and 185.9 mg/g DHA, while the isolated PL contained 35.2 mg/g EPAand 218.6 mg/g DHA. The total fatty acid profiles of the biomass,extracted crude oil, TAG fraction, and PL fraction are shown below inTable 26 and Table 27 calculated as mg/g and % FAME, respectively.

TABLE 26 Fatty Acid Profile of ATCC 20889 Calculated as Milligrams perGram Biomass Crude Oil TAG PL FAME FAME FAME FAME Fatty Acid (mg/g)(mg/g) (mg/g) (mg/g) C12:0* 0.0 0.0 0.0 0.0 C14:0* 0.5 8.3 32.0 2.8C14:1* 0.0 0.0 0.0 0.0 C15:0 0.0 0.0 0.0 0.1 C16:0* 6.6 80.3 150.8 50.4C16:1* 0.1 1.7 12.3 0.0 C18:0* 0.3 4.8 9.7 1.6 C18:1 N9* 0.6 6.6 7.6 1.2C18:1 N7 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 0.4 1.1 0.1 C20:0* 0.0 0.1 2.10.0 C18:3 N3* 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.00.0 0.0 C20:2 N6* 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.1 0.7 0.1 C22:0* 0.00.0 0.1 0.0 C20:4 N7 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 C20:4N6*0.4 1.4 1.1 7.2 C22:1 N9* 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 C20:4N3 0.0 0.5 0.9 0.5 C20:5 N3* 2.3 26.8 7.3 35.2 C24:0* 0.1 0.4 1.8 0.0C22:4 N9 0.0 3.4 0.2 2.8 C24:1 N9* 0.0 0.0 0.8 0.3 C22:5 N6* 5.0 63.676.2 45.9 C22:5 N3* 0.2 1.8 2.2 2.0 C22:6 N3* 16.5 205.1 185.9 218.6 Sumof all 32.8 405.2 493.0 368.7 FAME

TABLE 27 Fatty Acid Profiles of ATCC 20889 Calculated as a Percent ofTotal FAME Biomass Crude Oil TAG PL Fatty Acid % FAME % FAME % FAME %FAME C12:0* 0.0 0.0 0.0 0.0 C14:0* 1.4 2.0 6.5 0.8 C14:1* 0.0 0.0 0.00.0 C15:0 0.0 0.0 0.0 0.0 C16:0* 20.3 19.8 30.6 13.7 C16:1* 0.4 0.4 2.50.0 C18:0* 0.8 1.2 2.0 0.4 C18:1 N9* 1.8 1.6 1.5 0.3 C18:1 N7 0.0 0.00.0 0.0 C18:2 N6* 0.1 0.1 0.2 0.0 C20:0* 0.0 0.0 0.4 0.0 C18:3 N3* 0.00.0 0.0 0.0 C20:1 N9* 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.0 C20:2 N6*0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.1 0.0 C22:0* 0.0 0.0 0.0 0.0 C20:4 N70.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 C20:4N6* 1.3 0.4 0.2 2.0 C22:1N9* 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 C20:4 N3 0.1 0.1 0.2 0.1C20:5 N3* 7.0 6.6 1.5 9.5 C24:0* 0.3 0.1 0.4 0.0 C22:4 N9 0.0 0.8 0.00.8 C24:1 N9* 0.0 0.0 0.2 0.1 C22:5 N6* 15.3 15.7 15.5 12.5 C22:5 N3*0.5 0.5 0.5 0.5 C22:6 N3* 50.2 50.6 37.7 59.3 Sum of FAME % 100.0 100.0100.0 100.0

ATCC 20892—The lipid content of the biomass was estimated to be 8.8% asthe sum of FAME, and the amount of crude oil obtained after solventextraction was 12.1% by weight, giving a 138% recovery of fat present inthe biomass. The EPA and DHA content of the biomass was determined to be8.3 mg/g and 43.3 mg/g, respectively. The extracted crude oil contained50.5 mg/g EPA and 260.1 mg/g DHA. The isolated TAG contained 98.7 mg/gEPA and 407.7 mg/g DHA, while the isolated PL contained 50.4 mg/g EPAand 243.12 mg/g DHA. The total fatty acid profiles of the biomass,extracted crude oil, TAG fraction, and PL fraction are shown below inTable 28 and Table 29 calculated as mg/g and % FAME, respectively.

TABLE 28 Fatty Acid Profile of ATCC 20892 Calculated as Milligrams perGram Biomass Crude Oil TAG PL FAME FAME FAME FAME Fatty Acid (mg/g)(mg/g) (mg/g) (mg/g) C12:0* 0.0 0.0 0.0 0.0 C14:0* 7.3 42.2 29.7 54.7C14:1* 0.0 0.0 0.0 0.2 C15:0 0.0 0.0 0.0 0.0 C16:0* 18.9 109.0 133.5116.7 C16:1* 0.0 0.8 3.5 0.2 C18:0* 0.0 2.6 9.1 0.7 C18:1 N9* 0.6 7.322.0 1.3 C18:1 N7 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 1.4 9.7 0.4 C20:0* 0.00.0 0.0 0.0 C18:3 N3* 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.0 0.0 0.0 C18:4 N30.0 0.6 0.0 0.1 C20:2 N6* 0.0 0.0 0.0 0.0 C20:3 N6 0.3 1.1 6.3 0.6C22:0* 0.0 0.0 0.0 0.0 C20:4 N7 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0C20:4N6* 1.3 7.6 15.8 7.8 C22:1 N9* 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.0 0.00.0 C20:4 N3 0.3 1.5 4.7 1.4 C20:5 N3* 8.3 49.6 97.0 49.8 C24:0* 0.0 0.00.0 0.0 C22:4 N9 0.2 0.0 0.0 0.1 C24:1 N9* 0.0 1.1 4.9 1.0 C22:5 N6* 2.816.1 25.3 15.8 C22:5 N3* 1.1 6.6 23.9 6.0 C22:6 N3* 43.4 254.3 398.3239.5 Sum of all 87.5 508.0 800.7 498.2 FAME

TABLE 29 Fatty Acid Profiles of ATCC 20892 Calculated as a Percent ofTotal FAME Biomass Crude Oil TAG PL Fatty Acid % FAME % FAME % FAME %FAME C12:0* 0.0 0.0 0.0 0.0 C14:0* 8.3 8.3 3.6 11.0 C14:1* 0.0 0.0 0.00.0 C15:0 0.0 0.0 0.0 0.0 C16:0* 21.6 21.4 16.4 23.4 C16:1* 0.0 0.2 0.50.0 C18:0* 0.0 0.5 1.1 0.1 C18:1 N9* 0.7 1.4 2.6 0.2 C18:1 N7 0.0 0.00.0 0.0 C18:2 N6* 0.0 0.2 1.1 0.1 C20:0* 0.0 0.1 0.6 0.0 C18:3 N3* 0.00.0 0.0 0.0 C20:1 N9* 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.1 0.0 0.0 C20:2 N6*0.0 0.0 0.0 0.0 C20:3 N6 0.3 0.2 0.7 0.1 C22:0* 0.0 0.0 0.0 0.0 C20:4 N71.9 0.8 1.6 0.2 C20:3 N3 0.0 0.0 0.0 0.0 C20:4N6* 1.4 1.5 1.9 1.6 C22:1N9* 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 C20:4 N3 0.3 0.3 0.5 0.3C20:5 N3* 9.5 9.7 11.9 10.0 C24:0* 0.0 0.0 0.0 0.0 C22:4 N9 0.2 0.0 0.00.0 C24:1 N9* 0.0 0.2 0.5 0.2 C22:5 N6* 3.2 3.1 3.0 3.2 C22:5 N3* 1.31.2 2.8 1.2 C22:6 N3* 49.6 49.8 49.0 48.0 Sum of FAME % 100.0 100.0100.0 100.0

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

1. A microbial oil comprising a triglyceride fraction of at least 70% byweight, wherein the docosahexaenoic acid content of the triglyceridefraction is at least 50% by weight, wherein the docosapentaenoic acidn-6 content of the triglyceride fraction is from 0.5% by weight to 6% byweight, and wherein the oil comprises 5% by weight or less ofheptadecanoic acid.
 2. The microbial oil of claim 1, further comprisingan arachidonic acid content of the triglyceride fraction of 1.5% or lessby weight.
 3. A microbial oil comprising a triglyceride fraction of atleast 70% by weight, wherein the docosahexaenoic acid content of thetriglyceride fraction is at least 40% by weight, wherein thedocosapentaenoic acid n-6 content of the triglyceride fraction is fromat least 0.5% by weight to 6% by weight, wherein the ratio ofdocosahexaenoic acid to docosapentaenoic acid n-6 is greater than 6:1,and wherein the oil comprises 5% by weight or less of heptadecanoicacid.
 4. A microbial oil comprising a triglyceride fraction of at least70% by weight, wherein the docosahexaenoic acid content of thetriglyceride fraction is at least 60% by weight, and wherein the oilcomprises 5% by weight or less of heptadecanoic acid.
 5. The microbialoil of any one of claims 1 to 4, wherein at least 20% of thetriglycerides in the triglyceride fraction contain docosahexaenoic acidat two positions in the triglyceride selected from any two of the sn-1,sn-2, and sn-3 positions.
 6. The microbial oil of any one of claims 1 to4, wherein at least 5% of the triglycerides in the triglyceride fractioncontain docosahexaenoic acid at all three of the sn-1, sn-2, and sn-3positions in the triglyceride.
 7. A food product, cosmetic, orpharmaceutical composition for animals or humans, comprising themicrobial oil of any one of claims 1 to
 4. 8. The food product of claim7, wherein the food product is an infant formula.
 9. The food product ofclaim 8, wherein the infant formula can be consumed by prematureinfants.
 10. The food product of claim 7, wherein the food product ismilk, a beverage, a therapeutic drink, a nutritional drink, or acombination thereof.
 11. The food product of claim 7, wherein the foodproduct is an additive for animal or human food.
 12. The food product ofclaim 7, wherein the food product is a nutritional supplement.
 13. Thefood product of claim 7, wherein the food product is an animal feed. 14.The animal feed of claim 13, wherein the animal feed is an aquaculturefeed.
 15. The animal feed of claim 13, wherein the animal feed is adomestic animal feed, a zoological animal feed, a work animal feed, alivestock feed, or a combination thereof.